The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position.

Note: Low resolution figures with captions are embedded within the main text. High resolution figures are at the end of the main text and preceded by their captions.

Computationally efficient evaluation of penalized estimators of multivariate exponential family distributions is sought. These distributions encompass among others Markov random fields with variates of mixed type (e.g., binary and continuous) as special case of interest. The model parameter is estimated by maximization of the pseudo-likelihood augmented with a convex penalty. The estimator is shown to be consistent. With a world of multi-core computers in mind, a computationally efficient parallel Newton–Raphson algorithm is presented for numerical evaluation of the estimator alongside conditions for its convergence. Parallelization comprises the division of the parameter vector into subvectors that are estimated simultaneously and subsequently aggregated to form an estimate of the original parameter. This approach may also enable efficient numerical evaluation of other high-dimensional estimators. The performance of the proposed estimator and algorithm are evaluated and compared in a simulation study. Finally, the presented methodology is applied to data of an integrative omics study.

Determining whether life can progress arbitrarily slowly may reveal fundamental barriers to staying out of thermal equilibrium for living systems. By monitoring budding yeast’s slowed-down life at frigid temperatures and with modeling, we establish that Reactive Oxygen Species (ROS) and a global gene-expression speed quantitatively determine yeast’s pace of life and impose temperature-dependent speed limits - shortest and longest possible cell-doubling times. Increasing cells’ ROS concentration increases their doubling time by elongating the cell-growth (G1-phase) duration that precedes the cell-replication (S-G2-M) phase. Gene-expression speed constrains cells’ ROS-reducing rate and sets the shortest possible doubling-time. To replicate, cells require below-threshold concentrations of ROS. Thus, cells with sufficiently abundant ROS remain in G1, become unsustainably large and, consequently, burst. Therefore, at a given temperature, yeast’s replicative life cannot progress arbitrarily slowly and cells with the lowest ROS-levels replicate most rapidly. Fundamental barriers may constrain the thermal slowing of other organisms’ lives.

Life on Earth, including for microbes and cold-blooded animals, often occurs in frigid environments. At frigid temperatures, nearly all intracellular processes slow down which is colloquially said to decelerate life's pace and, potentially, aging. But even for one cell, an outstanding conceptual challenge is rigorously explaining how the slowed-down intracellular processes collectively sustain a cell's life and set its pace. Here, by monitoring individual yeast cells for months at near-freezing temperatures, we show how global gene-expression dynamics and Reactive Oxygen Species (ROS) act together as the primary factors that dictate and constrain the pace at which a budding yeast's life can progresses in frigid environments. We discovered that yeast cells help each other in surviving and dividing at frigid temperatures. By investigating the underlying mechanism, involving glutathione secretion, we discovered that ROS is the primary determinant of yeast's ability to survive and divide at near-freezing temperatures. Observing days-to-months-long cell-cycle progression in individual cells revealed that ROS inhibits S-G2-M (replicative) phase while elongating G1 (growth) phase up to a temperature-dependent threshold duration, beyond which yeast cannot divide and bursts as an unsustainably large cell. We discovered that an interplay between global gene-expression speed and ROS sets the threshold G1-duration by measuring rates of genome-wide transcription and protein synthesis at frigid temperatures and then incorporating them into a mathematical model. The same interplay yields unbeatable "speed limits" for cell cycling - shortest and longest allowed doubling times - at each temperature. These results establish quantitative principles for engineering cold-tolerant microbes and reveal how frigid temperatures can fundamentally constrain microbial life and cell cycle at the systems-level.