Characterizing Binding Interactions That Are Essential for Selective Transport through the Nuclear Pore Complex.

Specific macromolecules are rapidly transported across the nuclear envelope via the nuclear pore complex (NPC). The selective transport process is facilitated when nuclear transport receptors (NTRs) weakly and transiently bind to intrinsically disordered constituents of the NPC, FG Nups. These two types of proteins help maintain the selective NPC barrier. To interrogate their binding interactions in vitro, we deployed an NPC barrier mimic. We created the stationary phase by covalently attaching fragments of a yeast FG Nup called Nsp1 to glass coverslips. We used a tunable mobile phase containing NTR, nuclear transport factor 2 (NTF2). In the stationary phase, three main factors affected binding: the number of FG repeats, the charge of fragments, and the fragment density. We also identified three main factors affecting binding in the mobile phase: the avidity of the NTF2 variant for Nsp1, the presence of nonspecific proteins, and the presence of additional NTRs. We used both experimentally determined binding parameters and molecular dynamics simulations of Nsp1FG fragments to create an agent-based model. The results suggest that NTF2 binding is negatively cooperative and dependent on the density of Nsp1FG molecules. Our results demonstrate the strengths of combining experimental and physical modeling approaches to study NPC-mediated transport.

FG nucleoporins feature unique patterns that distinguish them from other IDPs.

FG nucleoporins (FG Nups) are intrinsically disordered proteins and are the putative regulators of nucleocytoplasmic transport. They allow fast, yet selective, transport of molecules through the nuclear pore complex, but the underlying mechanism of nucleocytoplasmic transport is not yet fully discovered. As a result, FG Nups have been the subject of extensive research in the past two decades. Although most studies have been focused on analyzing the conformation and function of FG Nups from a biophysical standpoint, some recent studies have investigated the sequence-function relationship of FG Nups, with a few investigating amino acid sequences of a large number of FG Nups to understand common characteristics that might enable their function. Previously, we identified an evolutionarily conserved feature in FG Nup sequences, which are extended subsequences with low charge density, containing only positive charges, and located toward the N-terminus of FG Nups. We named these patterns longest positive like charge regions (lpLCRs). These patterns are specific to positively charged residues, and negatively charged residues do not demonstrate such a pattern. In this study, we compare FG Nups with other disordered proteins obtained from the DisProt and UniProt database in terms of presence of lpLCRs. Our results show that the lpLCRs are virtually exclusive to FG Nups and are not observed in other disordered proteins. Also, lpLCRs are what differentiate FG Nups from DisProt proteins in terms of charge distribution, meaning that excluding lpLCRs from the sequences of FG Nups make them similar to DisProt proteins in terms of charge distribution. We also previously showed the biophysical effect of lpLCRs in conformation of FG Nups. The results of this study are in line with our previous findings and imply that lpLCRs are virtually exclusive and functionally significant characteristics of FG Nups and nucleocytoplasmic transport.

Agent-Based Modeling in Molecular Systems Biology.

Molecular systems orchestrating the biology of the cell typically involve a complex web of interactions among various components and span a vast range of spatial and temporal scales. Computational methods have advanced our understanding of the behavior of molecular systems by enabling us to test assumptions and hypotheses, explore the effect of different parameters on the outcome, and eventually guide experiments. While several different mathematical and computational methods are developed to study molecular systems at different spatiotemporal scales, there is still a need for methods that bridge the gap between spatially-detailed and computationally-efficient approaches. In this review, we summarize the capabilities of agent-based modeling (ABM) as an emerging molecular systems biology technique that provides researchers with a new tool in exploring the dynamics of molecular systems/pathways in health and disease.

Quality control of mRNAs at the entry of the nuclear pore: Cooperation in a complex molecular system.

Despite extensive research on how mRNAs are quality controlled prior to export into the cytoplasm, the exact underlying mechanisms are still under debate. Specifically, it is unclear how quality control proteins at the entry of the nuclear pore complex (NPC) distinguish normal and aberrant mRNAs. While some of the involved components are suggested to act as switches and recruit different factors to normal versus aberrant mRNAs, some experimental and computational evidence suggests that the combined effect of the regulated stochastic interactions between the involved components could potentially achieve an efficient quality control of mRNAs. In this review, we present a state-of-the-art portrait of the mRNA quality control research and discuss the current hypotheses proposed for dynamics of the cooperation between the involved components and how it leads to their shared goal: mRNA quality control prior to export into the cytoplasm.

Looking "Under the Hood" of Cellular Mechanotransduction with Computational Tools: A Systems Biomechanics Approach across Multiple Scales.

Signal modulation has been developed in living cells throughout evolution to promote utilizing the same machinery for multiple cellular functions. Chemical and mechanical modules of signal transmission and transduction are interconnected and necessary for organ development and growth. However, due to the high complexity of the intercommunication of physical intracellular connections with biochemical pathways, there are many missing details in our overall understanding of mechanotransduction processes, i.e., the process by which mechanical signals are converted to biochemical cascades. Cell-matrix adhesions are mechanically coupled to the nucleus through the cytoskeleton. This modulated and tightly integrated network mediates the transmission of mechanochemical signals from the extracellular matrix to the nucleus. Various experimental and computational techniques have been utilized to understand the basic mechanisms of mechanotransduction, yet many aspects have remained elusive. Recently, in silico experiments have made important contributions to the field of mechanobiology. Herein, computational modeling efforts devoted to understanding integrin-mediated mechanotransduction pathways are reviewed, and an outlook is presented for future directions toward using suitable computational approaches and developing novel techniques for addressing important questions in the field of mechanotransduction.

The LINC and NPC relationship - it's complicated!

The genetic information of eukaryotic cells is enclosed within a double-layered nuclear envelope, which comprises an inner and outer nuclear membrane. Several transmembrane proteins locate to the nuclear envelope; however, only two integral protein complexes span the nuclear envelope and connect the inside of the nucleus to the cytoplasm. The nuclear pore complex (NPC) acts as a gateway for molecular exchange between the interior of the nucleus and the cytoplasm, whereas so-called LINC complexes physically link the nucleoskeleton and the cytoskeleton. In this Commentary, we will discuss recent studies that have established direct functional associations between these two complexes. The assembly of NPCs and their even distribution throughout the nuclear envelope is dependent on components of the LINC complex. Additionally, LINC complex formation is dependent on the successful localization of inner nuclear membrane components of LINC complexes and their transport through the NPC. Furthermore, the architecture of the nuclear envelope depends on both protein complexes. Finally, we will present recent evidence showing that LINC complexes can affect nucleo-cytoplasmic transport through the NPC, further highlighting the importance of understanding the associations of these essential complexes at the nuclear envelope.

Nucleoporin's Like Charge Regions Are Major Regulators of FG Coverage and Dynamics Inside the Nuclear Pore Complex.

Nucleocytoplasmic transport has been the subject of a large body of research in the past few decades. Recently, the focus of investigations in this field has shifted from studies of the overall function of the nuclear pore complex (NPC) to the examination of the role of different domains of phenylalanine-glycine nucleoporin (FG Nup) sequences on the NPC function. In our recent bioinformatics study, we showed that FG Nups have some evolutionarily conserved sequence-based features that might govern their physical behavior inside the NPC. We proposed the 'like charge regions' (LCRs), sequences of charged residues with only one type of charge, as one of the features that play a significant role in the formation of FG network inside the central channel. In this study, we further explore the role of LCRs in the distribution of FG Nups, using a recently developed coarse-grained molecular dynamics model. Our results demonstrate how LCRs affect the formation of two transport pathways. While some FG Nups locate their FG network at the center of the NPC forming a homogeneous meshwork of FG repeats, other FG Nups cover the space adjacent to the NPC wall. LCRs in the former group, i.e. FG Nups that form an FG domain at the center, tend to regulate the size of the highly dense, doughnut-shaped FG meshwork and leave a small low FG density area at the center of the pore for passive diffusion. On the other hand, LCRs in the latter group of FG Nups enable them to maximize their interactions and cover a larger space inside the NPC to increase its capability to transport numerous cargos at the same time. Finally, a new viewpoint is proposed that reconciles different models for the nuclear pore selective barrier function.

Torsional behavior of axonal microtubule bundles.

Axonal microtubule (MT) bundles crosslinked by microtubule-associated protein (MAP) tau are responsible for vital biological functions such as maintaining mechanical integrity and shape of the axon as well as facilitating axonal transport. Breaking and twisting of MTs have been previously observed in damaged undulated axons. Such breaking and twisting of MTs is suggested to cause axonal swellings that lead to axonal degeneration, which is known as "diffuse axonal injury". In particular, overstretching and torsion of axons can potentially damage the axonal cytoskeleton. Following our previous studies on mechanical response of axonal MT bundles under uniaxial tension and compression, this work seeks to characterize the mechanical behavior of MT bundles under pure torsion as well as a combination of torsional and tensile loads using a coarse-grained computational model. In the case of pure torsion, a competition between MAP tau tensile and MT bending energies is observed. After three turns, a transition occurs in the mechanical behavior of the bundle that is characterized by its diameter shrinkage. Furthermore, crosslink spacing is shown to considerably influence the mechanical response, with larger MAP tau spacing resulting in a higher rate of turns. Therefore, MAP tau crosslinking of MT filaments protects the bundle from excessive deformation. Simultaneous application of torsion and tension on MT bundles is shown to accelerate bundle failure, compared to pure tension experiments. MAP tau proteins fail in clusters of 10-100 elements located at the discontinuities or the ends of MT filaments. This failure occurs in a stepwise fashion, implying gradual accumulation of elastic tensile energy in crosslinks followed by rupture. Failure of large groups of interconnecting MAP tau proteins leads to detachment of MT filaments from the bundle near discontinuities. This study highlights the importance of torsional loading in axonal damage after traumatic brain injury.

Buckling behavior of individual and bundled microtubules.

As the major structural constituent of the cytoskeleton, microtubules (MTs) serve a variety of biological functions that range from facilitating organelle transport to maintaining the mechanical integrity of the cell. Neuronal MTs exhibit a distinct configuration, hexagonally packed bundles of MT filaments, interconnected by MT-associated protein (MAP) tau. Building on our previous work on mechanical response of axonal MT bundles under uniaxial tension, this study is focused on exploring the compression scenarios. Intracellular MTs carry a large fraction of the compressive loads sensed by the cell and therefore, like any other column-like structure, are prone to substantial bending and buckling. Various biological activities, e.g., actomyosin contractility and many pathological conditions are driven or followed by bending, looping, and buckling of MT filaments. The coarse-grained model previously developed in our lab has been used to study the mechanical behavior of individual and bundled in vivo MT filaments under uniaxial compression. Both configurations show tip-localized, decaying, and short-wavelength buckling. This behavior highlights the role of the surrounding cytoplasm and MAP tau on MT buckling behavior, which allows MT filaments to bear much larger compressive forces. It is observed that MAP tau interconnections improve this effect by a factor of two. The enhanced ability of MT bundles to damp buckling waves relative to individual MT filaments, may be interpreted as a self-defense mechanism because it helps axonal MTs to endure harsher environments while maintaining their function. The results indicate that MT filaments in a bundle do not buckle simultaneously implying that the applied stress is not equally shared among the MT filaments, that is a consequence of the nonuniform distribution of MAP tau proteins along the bundle length. Furthermore, from a pathological perspective, it is observed that axonal MT bundles are more vulnerable to failure in compression than tension.