The Role of FG Nucleoporins Amino Acid Sequence Composition in Nucleocytoplasmic Transport

Bidirectional transport of molecules through the nuclear envelope (nucleocytoplasmic transport) is a vital, yet very complex process inside eukaryotic cells. This process is facilitated by thousands of nanoscale pores embedded in the nuclear envelope, named nuclear pore complexes (NPCs). The remarkable feature of NPC is that it facilitates transport in a fast, yet selective manner. Despite numerous studies, the underlying mechanism of NPC’s function is unknown. The intricate function of NPC cannot be fully captured via experimental techniques due to small scale and rapid movement of the FG Nucleoporins, which are intrinsically disordered proteins rich in phenylalanine-glycine repeats, responsible for facilitating the transport process. In this dissertation, using a range of computational approaches, the role of FG Nups in the nuclear transport process is explored. FG Nups do not have a well-defined secondary structure and it is believed that their sequence composition and conformational ensemble are critical for their function in the NPC. In this dissertation, for the first time, specific sequence patterns in the charge distribution of FG Nups were identified that were not observed in other intrinsically disordered proteins. These patterns are extended sub-sequences that only contain positively charged residues, have low charge density, and are located toward the N-terminus of FG Nups. We named these evolutionarily conserved patterns like charge regions (LCRs). Additionally, the role that LCRs play in the conformational ensemble and function of FG Nups was examined, using coarse-grained molecular dynamics simulations. Our simulations in multiple levels (single Nups, ring cross sections of NPC, and whole NPC) show that number of charged residues in the LCR impact the conformational ensemble of FG Nups and movement of the cargo complex. Our simulations also suggest that the number of charged residues in LCR can regulate the interaction of cargo complex with FG Nups. Since conformational ensemble of FG Nups and their interaction with cargo complex are two governing factors of the transport process, we suggest that LCRs, the unique and evolutionarily conserved features of FG Nups, are major regulators of the nucleocytoplasmic transport

The Role of FG Nucleoporins Amino Acid Sequence Composition in Nucleocytoplasmic Transport

Bidirectional transport of molecules through the nuclear envelope (nucleocytoplasmic transport) is a vital, yet very complex process inside eukaryotic cells. This process is facilitated by thousands of nanoscale pores embedded in the nuclear envelope, named nuclear pore complexes (NPCs). The remarkable feature of NPC is that it facilitates transport in a fast, yet selective manner. Despite numerous studies, the underlying mechanism of NPC’s function is unknown. The intricate function of NPC cannot be fully captured via experimental techniques due to small scale and rapid movement of the FG Nucleoporins, which are intrinsically disordered proteins rich in phenylalanine-glycine repeats, responsible for facilitating the transport process. In this dissertation, using a range of computational approaches, the role of FG Nups in the nuclear transport process is explored. FG Nups do not have a well-defined secondary structure and it is believed that their sequence composition and conformational ensemble are critical for their function in the NPC. In this dissertation, for the first time, specific sequence patterns in the charge distribution of FG Nups were identified that were not observed in other intrinsically disordered proteins. These patterns are extended sub-sequences that only contain positively charged residues, have low charge density, and are located toward the N-terminus of FG Nups. We named these evolutionarily conserved patterns like charge regions (LCRs). Additionally, the role that LCRs play in the conformational ensemble and function of FG Nups was examined, using coarse-grained molecular dynamics simulations. Our simulations in multiple levels (single Nups, ring cross sections of NPC, and whole NPC) show that number of charged residues in the LCR impact the conformational ensemble of FG Nups and movement of the cargo complex. Our simulations also suggest that the number of charged residues in LCR can regulate the interaction of cargo complex with FG Nups. Since conformational ensemble of FG Nups and their interaction with cargo complex are two governing factors of the transport process, we suggest that LCRs, the unique and evolutionarily conserved features of FG Nups, are major regulators of the nucleocytoplasmic transport

Nucleoporins' exclusive amino acid sequence features regulate their transient interaction with and selectivity of cargo complexes in the nuclear pore.

Nucleocytoplasmic traffic of nucleic acids and proteins across the nuclear envelop via the nuclear pore complexes (NPCs) is vital for eukaryotic cells. NPCs screen transported macromolecules based on their morphology and surface chemistry. This selective nature of the NPC-mediated traffic is essential for regulating the fundamental functions of the nucleus, such as gene regulation, protein synthesis, and mechanotransduction. Despite the fundamental role of the NPC in cell and nuclear biology, the detailed mechanisms underlying how the NPC works have remained largely unknown. The critical components of NPCs enabling their selective barrier function are the natively unfolded phenylalanine- and glycine-rich proteins called "FG-nucleoporins" (FG Nups). These intrinsically disordered proteins are tethered to the inner wall of the NPC, and together form a highly dynamic polymeric meshwork whose physicochemical conformation has been the subject of intense debate. We observed that specific sequence features (called largest positive like-charge regions, or lpLCRs), characterized by extended subsequences that only possess positively charged amino acids, significantly affect the conformation of FG Nups inside the NPC. Here we investigate how the presence of lpLCRs affects the interactions between FG Nups and their interactions with the cargo complex. We combine coarse-grained molecular dynamics simulations with time-resolved force distribution analysis to disordered proteins to explore the behavior of the system. Our results suggest that the number of charged residues in the lpLCR domain directly governs the average distance between Phe residues and the intensity of interaction between them. As a result, the number of charged residues within lpLCR determines the balance between the hydrophobic interaction and the electrostatic repulsion and governs how dense and disordered the hydrophobic network formed by FG Nups is. Moreover, changing the number of charged residues in an lpLCR domain can interfere with ultrafast and transient interactions between FG Nups and the cargo complex

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

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

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

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