Journal: Science Advances

Article Title: Bridging-induced phase separation induced by cohesin SMC protein complexes

doi: 10.1126/sciadv.abe5905

Figure Lengend Snippet: ( A ) Snapshots before and after cohesin-induced compaction of a doubly tethered DNA molecule. In schematics on the right, blue represents DNA, yellow represents biotin-streptavidin, and red represents the cohesin holocomplex. ( B ) Snapshots and schematics of side-flow experiment before and after addition of cohesin complexes. Yellow arrow indicates a DNA region that is tightly clustered (i.e., not a DNA loop). ( C ) DNA cluster size as a function of time (right) calculated from the integrated fluorescence intensities in the cluster and the full 48.5-kbp λDNA in the image (left). Yellow circle indicates the compaction spot. ( D ) DNA compaction time under various conditions ( n = 33, 25, 10, 13, 10, 13, 7, 11, and 9, respectively). ( E ) Cluster formation with labeled cohesin holocomplex showing the colocalization of compacted DNA (blue) and cohesin (red). ( F ) Representative measured trace of DNA compaction. ( G ) Simultaneously measured cohesin binding trace. ( H ) Histogram of bleaching step intensities of single Alexa647-cohesin molecules ( n = 64). Inset shows a representative bleaching trace of a small cluster. A Gaussian fit (blue) yielded 3.0 ± 1.2 arbitrary units (a.u.) (means ± SD). ( I ) Number of cohesin holocomplexes in a cluster. A Gaussian fit (blue) yielded 740 ± 500 (means ± SD). ( J to L ) Atomic force microscopy (AFM) images of DNA/cohesin-holocomplex, cohesin-holocomplex only, and DNA only, respectively. ( M ) Volumes of the DNA/cohesin clusters for different conditions (median ± SEM; n = 21, 11, and 57). Red line in inset illustrates a cluster with its boundary (red). Two-paired Student’s t test was used for (D) and (M).

Article Snippet: Concentrations of λDNA (100 ng/μl; Promega) and 50 nM cohesin with or without 50 nM loader were used.

Techniques: Fluorescence, Labeling, Binding Assay, Microscopy

Journal: Science Advances

Article Title: Bridging-induced phase separation induced by cohesin SMC protein complexes

doi: 10.1126/sciadv.abe5905

Figure Lengend Snippet: ( A ) Images of a DNA/cohesin-holocomplex droplet (top left) and 20-nm QD (top right). White dashed lines indicate where cross-sectional intensity profiles were acquired, yielding the bottom panels with Gaussian fits. ( B ) Diameter distributions of cohesin droplets (means ± SD, 1.14 ± 0.18 μm; n = 151) and QDs (means ± SD, 0.57 ± 0.11 μm; n = 59). ( C ) Circularity distributions of cohesin droplets ( n = 57). ( D ) Cohesin holocomplex (red) forms liquid-phase droplets along a DNA (green). Over time, two droplets are seen to fuse into one spherical droplet (movie S4). ( E ) Merging of two clusters, as monitored in the cohesin (top) and DNA (bottom) channels. Left shows three snapshots; right shows fluorescence intensity kymographs ( n = 18). ( F ) Fluorescence recovery after photobleaching (FRAP) experiment of cohesin droplet. Snapshot images of a cohesin droplet on DNA, before and after photobleaching and after recovery (top). Cohesin cluster intensity versus time (bottom; mean ± SEM, n = 44). Time constant is 126 ± 4 s (error is SD).

Article Snippet: Concentrations of λDNA (100 ng/μl; Promega) and 50 nM cohesin with or without 50 nM loader were used.

Techniques: Fluorescence

Journal: Science Advances

Article Title: Bridging-induced phase separation induced by cohesin SMC protein complexes

doi: 10.1126/sciadv.abe5905

Figure Lengend Snippet: ( A ) Phase diagram of cluster formation induced by cohesin holocomplex and λDNA at various salt (NaCl) and cohesin-holocomplex concentrations. Red squares indicate that DNA compaction occurred, while blue circles indicate that this did not occur. Solid line is a guide to the eye. ( B ) Reversibility test with washing in a high-salt buffer. Cohesin cluster intensities versus time upon washing with buffers with different salt concentrations (bottom; errors are SD). Dissociation times (τ) are 476 ± 25 s, 41.9 ± 0.7 s, 7.5 ± 0.2 s, and 0.79 ± 0.03 s (errors are SD) for bleaching, 50, 100, and 500 mM NaCl, respectively ( n = 60, 113, 93, and 150). ( C ) Dissolving of a cohesin cluster by washing with 1,6-hexanediol. Cohesin-cluster intensity versus time upon washing with 1,6-hexanediol (bottom). τ = 476 ± 25 s, 7.9 ± 0.1 s, and 2.2 ± 0.1 s for bleaching, 5 and 10% 1,6-hexanediol buffer, respectively ( n = 60, 80, and 37). ( D ) Scheme for in vivo 1,6-hexanediol treatment of yeast cells. ( E ) Chromatin immunoprecipitation–quantitative real-time polymerase chain reaction (ChIP-qPCR) results for cohesin (Smc3-Pk) and binding to a centromere ( CEN3 ), chromosome arm ( POA1 ), and a negative-control binding site ( TUB2 ), before (Pre) and after 1,6-hexanediol (Hex) treatment and after washing out 1,6-hexanediol (Rec) (means ± SEM, n = 3). Tetracyclin repressor–green fluorescent protein (TetR-GFP) binding was analyzed as a control. ( F ) Same for binding to tetracyclin operator (TetO) sites (means ± SEM, n = 3).

Article Snippet: Concentrations of λDNA (100 ng/μl; Promega) and 50 nM cohesin with or without 50 nM loader were used.

Techniques: In Vivo, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Negative Control, Control

Journal: Science Advances

Article Title: Bridging-induced phase separation induced by cohesin SMC protein complexes

doi: 10.1126/sciadv.abe5905

Figure Lengend Snippet: ( A to D ) Representative AFM images of cohesin holocomplex as it binds to different DNA lengths of 100 bp, 3 kbp, 5 kbp, and 48.5 kbp, respectively. ( E ) Radius of gyration of cohesin-DNA clusters versus DNA length (median ± SEM). Note the log-log scale. At low DNA length l , the cluster size is constant (green line), while beyond a critical length l C ≈ 3 kbp, the data exhibit a power law, R G ~ l α (blue line) with α = 0.45 ± 0.01 (SD) from a fit. Inset shows data from molecular dynamics (MD) simulations for cohesin with biconnectivity (black, α = 0.45 ± 0.01), triconnectivity (violet, α = 0.40 ± 0.01), and multiconnectivity (green, α = 0.33 ± 0.01). ( F ) Examples of single cohesin holocomplex bound to 100-bp DNA. ( G ) Representative image of DNA bridging by a single cohesin holocomplex. ( H ) Probability that a single cohesin holocomplex that bound DNA did or did not bridge to another segment along the DNA ( n = 81). ( I ) Working BIPS model for cohesin-mediated phase separation. Bare DNA is bridged by a single cohesin holocomplex, increasing the local concentration of DNA, which subsequently induces binding of more cohesin holocomplexes to this region, leading to the formation of a large DNA/cohesin-holocomplex droplet. The cartoons are not drawn to scale.

Article Snippet: Concentrations of λDNA (100 ng/μl; Promega) and 50 nM cohesin with or without 50 nM loader were used.

Techniques: Concentration Assay, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Chick Ciliary Ganglion Neurons Contain Transcripts Coding for Acetylcholine Receptor-Associated Protein at Synapses (Rapsyn)

doi: 10.1523/JNEUROSCI.17-13-05016.1997

Figure Lengend Snippet: Amplification of rapsyn-like cDNAs from chick muscle and neuronal tissue templates. A, PCR products detected with ethidium bromide staining. Control template reactions (lanes 2, 3) revealed single products of ≈1470 or 1350 bp after amplification of Ch43K.1 insert using primer pairs S1/A1 or S2/A2, respectively. Subsequent test reactions (lanes 4–9) using only the S1/A1 primer pair reveal products amplified from muscle (Mu), ciliary ganglion (CG), or brain (Br) cDNA templates, synthesized by reverse transcription of cellular RNA (+; lanes 4, 6, 8). Note that multiple products are amplified from muscle cDNA templates (∼1500, 1350, 1150, and 500 bp; lane 4), whereas only a 500 bp product is amplified from brain and ganglionic templates (lanes 6, 8). Negative controls, using “sham” reverse transcriptions from corresponding muscle, brain, and CG reactions lacking RT (−; lanes 5, 7,9), revealed no products. pGEM DNA marker was loaded inlanes 1 and 10, and sizes are labeled atleft. B, Tissue PCR products amplified as in A were detected by Southern blot hybridization using the 32-P-labeled ChIN probe (see Materials and Methods; Fig. ​Fig.22B). Autoradiograms exposed for 1 hr from reactions using muscle templates (lane 1) detected the three cDNA products (∼1500, 1350, 1150) resolved on ethidium-stained gels (compare with A) as well as a less intense product at 1000 bp. Subsequent lanes (3–10) depict PCR products amplified from CG, Br, DRG, and liver (Lv) templates exposed for 10–24 hr. Note that the same two product sizes seen for Mu reactions (∼1500 and 1350 bp) are also resolved in CG and Br reactions (lanes 3,5) and that an additional product of 1600 bp is detected in CG. No products are detected in amplifications from DRG (lane 7) or Lv (lane 9) templates, even after exposures >36 hr. As in A, + (lanes 1,3, 5, 7, 9) and − (lanes 2, 4,6, 8, 10) denote presence or absence, respectively, of RT from the initial reverse transcription reaction. Lane 11 depicts the migration of the 292 bp32-P-labeled ChIN probe. Size markers are pGEM standards (in base pairs) reproduced from the original agarose gel (data not shown).

Article Snippet: The second strands were synthesized from the single-strand cDNA templates using Escherichia coli λDNA polymerase I (500 U/ml, Promega) in the presence of E. coli ribonuclease H (20 U/ml, Promega) for 1 hr at 12°C and 1 hr at 21°C, and the ends blunted with T4 DNA polymerase (40 U/ml, Promega) for 1 hr at 37°C.

Techniques: Amplification, Staining, Control, Synthesized, Reverse Transcription, Marker, Labeling, Southern Blot, Hybridization, Migration, Agarose Gel Electrophoresis