Single cohesin molecules generate force by two distinct mechanisms

Spatial organization of DNA is facilitated by cohesin protein complexes that move on DNA and extrude DNA loops. How cohesin works mechanistically as a molecular machine is poorly understood. Here, we measure mechanical forces generated by conformational changes in single cohesin molecules. We show that bending of SMC coiled coils is driven by random thermal fluctuations leading to a ~32 nm head-hinge displacement that resists forces up to 1 pN; ATPase head engagement occurs in a single step of ~10 nm and is driven by an ATP dependent head-head movement, resisting forces up to 15 pN. Our molecular dynamic simulations show that the energy of head engagement can be stored in a mechanically strained conformation of NIPBL and released during disengagement. These findings reveal how single cohesin molecules generate force by two distinct mechanisms. We present a model, which proposes how this ability may power different aspects of cohesin-DNA interaction

Mechanical disengagement of the cohesin ring

Cohesin forms a proteinaceous ring that is thought to link sister chromatids by entrapping DNA and counteracting the forces generated by the mitotic spindle. Whether individual cohesins encircle both sister DNAs and how cohesin opposes spindle-generated forces remains unknown. Here we perform force measurements on individual yeast cohesin complexes either bound to DNA or holding together two DNAs. By covalently closing the hinge and Smc3Psm3–kleisin interfaces we find that the mechanical stability of the cohesin ring entrapping DNA is determined by the hinge domain. Forces of ~20 pN disengage cohesin at the hinge and release DNA, indicating that ~40 cohesin molecules are sufficient to counteract known spindle forces. Our findings provide a mechanical framework for understanding how cohesin interacts with sister chromatids and opposes the spindle-generated tension during mitosis, with implications for other force-generating chromosomal processes including transcription and DNA replication

Direct detection of a single photon by humans

Despite investigations for over 70 years, the absolute limits of human vision have remained unclear. Rod cells respond to individual photons, yet whether a single-photon incident on the eye can be perceived by a human subject has remained a fundamental open question. Here we report that humans can detect a single-photon incident on the cornea with a probability significantly above chance. This was achieved by implementing a combination of a psychophysics procedure with a quantum light source that can generate single-photon states of light. We further discover that the probability of reporting a single photon is modulated by the presence of an earlier photon, suggesting a priming process that temporarily enhances the effective gain of the visual system on the timescale of seconds

Rapid movement and transcriptional re-localization of human cohesin on DNA

The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion

Model of a depolymerizing microtubule [Microtubule depolymerization as a biological machine]

This video shows the shortening plus end of a microtubule polymer with 13 protofilaments, which are arranged in a 3-start left handed helix (the most common configuration in cells). Tubulin dimers, consisting of alpha-tubulin (dark green) and beta-tubulin (light green), form a hollow tube 25 nm in a diameter. In this molecular-mechanical model of a microtubule each tubulin interacts via defined energy relationships with its longitudinal and lateral neighbors. The calculations begin with an initial configuration in which all protofilaments are perfectly straight. However, the minimum energy configuration for each pair of longitudinally attached tubulins is when they form roughly 22 degree angle. As a result, each protofilament tends to curl and form a 'ram's horn'. Tubulin dimers begin to dissociate from the protofilaments ends soon after they loose their lateral bonds (in the model the dissociation of the terminal dimer takes place when it bends greater than 90 degrees away from the microtubule axis)Componente Curricular::Educação Superior::Ciências Biológicas::Morfologi

Model of a depolymerizing microtubule [Microtubule depolymerization as a biological machine]

This video shows the shortening plus end of a microtubule polymer with 13 protofilaments, which are arranged in a 3-start left handed helix (the most common configuration in cells). Tubulin dimers, consisting of alpha-tubulin (dark green) and beta-tubulin (light green), form a hollow tube 25 nm in a diameter. In this molecular-mechanical model of a microtubule each tubulin interacts via defined energy relationships with its longitudinal and lateral neighbors. The calculations begin with an initial configuration in which all protofilaments are perfectly straight. However, the minimum energy configuration for each pair of longitudinally attached tubulins is when they form roughly 22 degree angle. As a result, each protofilament tends to curl and form a 'ram's horn'. Tubulin dimers begin to dissociate from the protofilaments ends soon after they loose their lateral bonds (in the model the dissociation of the terminal dimer takes place when it bends greater than 90 degrees away from the microtubule axis)Componente Curricular::Educação Superior::Ciências Biológicas::Morfologi

A Molecular-Mechanical Model of the Microtubule

Dynamic instability of MTs is thought to be regulated by biochemical transformations within tubulin dimers that are coupled to the hydrolysis of bound GTP. Structural studies of nucleotide-bound tubulin dimers have recently provided a concrete basis for understanding how these transformations may contribute to MT dynamic instability. To analyze these ideas, we have developed a molecular-mechanical model in which structural and biochemical properties of tubulin are used to predict the shape and stability of MTs. From simple and explicit features of tubulin, we define bond energy relationships and explore the impact of their variations on integral MT properties. This modeling provides quantitative predictions about the GTP cap. It specifies important mechanical features underlying MT instability and shows that this property does not require GTP-hydrolysis to alter the strength of tubulin-tubulin bonds. The MT plus end is stabilized by at least two layers of GTP-tubulin subunits, whereas the minus end requires at least one; this and other differences between the ends are explained by asymmetric force balances. Overall, this model provides a new link between the biophysical characteristics of tubulin and the physiological behavior of MTs. It will also be useful in building a more complete description of MT dynamics and mechanics

An extrinsic motor directs chromatin loop formation by cohesin.

The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation