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.

Integrins are Alpha-Beta transmembrane receptors that mediate cell-­matrix and cell-­cell adhesion, comprised of multi-­domain, massive ectodomains, single-­pass, transmembrane domains, and short, floppy cytoplasmic domains. They link the extracellular matrix or counter-­receptors on other cells with the contractile cytoskeleton, mediating the transduction of mechanochemical signals across the plasma membrane and playing critical roles in a host of cellular functions, such as migration, cell traction, motility, platelet aggregation, and leukocyte transmigration.

Functionally, integrins are switch-­like proteins that can take on at least three different functional states: inactive, active, and ligand-bound. Integrin function is dependent upon allosteric conformational changes in its structure. Integrins are by default in an inactive (low affinity) state and can be activated via interacting with cytoplasmic proteins (e.g. talin) and/or engaging with extracellular ligands (e.g. fibrinogen). Integrin activation triggered by a cytoplasmic signal is called inside-­out signaling, while outside-­in signaling is defined as ligand-­integrin binding followed by conformational changes in the transmembrane and cytoplasmic domains. Association of the integrin with the ligand induces quaternary changes in the integrin, leading to cell signaling and dynamic cell adhesion. However, atomistic details of these conformational changes as well as mechanisms of integrin clustering are not fully understood.

This study employs molecular dynamics techniques to provide detailed, mechanistic answers for a few key questions on integrin (Alpha)IIb(Beta)3 function, a ­platelet-specific integrin member that plays a critical role in thrombosis. It is highly debated whether integrin transmembrane domains form homo-­oligomers, leading to focal adhesion growth. This study suggests that homo-­ oligomerization of the Beta subunit potentially regulates integrin clustering, as opposed to the Alpha subunit, which appears to be a poor regulator for the clustering process. Two distinct hypotheses are proposed to explain the atomic mechanism of integrin activation and how conformational changes triggered by cytoplasmic/extracellular proteins are propagated across the integrin structure: The switch-blade and the deadbolt model. To reconcile these apparently-contradictory models for integrin activation, this work investigated the mechanism of integrin (Alpha)IIb(Beta)3 inside-­out activation triggered by interactions with the cytoplasmic protein talin, and its outside-­in activation as a result of exposure to the soluble RGD ligand. Finally, it was shown that the integrin Alpha subunit head domain regulates integrin-­ligand binding affinity indirectly via inducing conformational changes in a key metal ion binding site (named LIMBS) in the Beta subunit head domain. Hence, it was concluded that different ligand binding affinities of integrin (Alpha)IIb(Beta)3 and (Alpha)V(Beta)3 is attributed to the larger attraction between the (Alpha)V subunit head domain and the metal ion binding site LIMBS.

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. Export and quality control of messenger ribonucleic acids (mRNAs) feature a prominent example of such an intricate molecular system. Export of mRNAs into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. 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 aberrant mRNAs are recognized and retained inside the nucleus. 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. In this dissertation, I present my research on mRNA quality control using different computational techniques.

Using the agent-based modeling (ABM) approach, which is an emerging molecular systems biology technique for exploring the dynamics of molecular systems/pathways in health and disease, we first developed a minimal model of the mRNA quality control (QC) mechanism. Our results suggested that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the RBPs and nuclear basket proteins, namely myosin-like protein-1 (Mlp1) or translocated promoter region (Tpr) protein, are the minimum requirements to distinguish and retain aberrant mRNAs. In addition, we demonstrated how the length of mRNA may affect the QC process.

The interaction between Mlp1 with one of the Saccharomyces cerevisiae RBPs, namely the nuclear polyadenylated RNA-binding protein (Nab2), was then investigated. Mlp1 plays a substantial role in mRNA quality control by interacting with other proteins involved in this process, specifically the RBPs. Yet, the mechanism of the interaction between Mlp1 and RBPs is still elusive. Using an array of integrated computational approaches including protein structure prediction, protein-protein docking, and molecular dynamics simulations, we dissected Mlp1-Nab2 interaction. Our results were consistent with experimental observations, which suggested that Nab2 residue F73 is essential for Mlp1 binding and further uncovered an indirect role of Nab2-F73 in this interaction.

Mechanical signals affect virtually every fundamental single- and multi-cellular process in biology. The local responses of individual molecules to mechanical stimuli at the interface of cell with its adjacent microenvironment (extracellular matrix or material) elicit global responses at the cell and tissue scales. Understanding and manipulating the cell-material interaction can be leveraged to design biomaterials with unique characteristics tailored towards a wide variety of biological applications such as platforms that direct stem cell differentiation for tissue engineering, sensors that can record accurate electrical signals in single cells for neuroscience, and implants that are susceptible to cell adhesion for biomedical applications. In this thesis I present work characterizing the response of cells to mechanical stimuli at the single cell and single molecule scales. At the single cell scale, we provide insights into how mechanical signals such as micro- and nano-topography of metallic and metallic surfaces affect cell adhesion, both in mammalian and bacterial cells. Next we characterize the mechanical response of protein complexes involved in the transmission of mechanical signals across the cytoskeleton to the nucleus.

The four main contributions of the work presented in this thesis are as follows: 1) We used high resolution scanning electron microscopy to characterize the cell-nanostructure interface and provide insights into the response of individual mammalian cells to nanostructures with complex geometries. 2) We provide a first look at how individual bacterial cells adhere to metallic nanostructures, which could lead to new techniques to thwart infections. 3) We proposed a novel technique to control the growth and arrangements of bacterial cell communities. This method will allow precise small-scale mechanical manipulation of bacterial cells and could be utilized for unraveling the understudied mechanisms of bacterial mechanosensitivity. 4) We performed the first molecular dynamics study on the mechanisms of force transmission to the nucleus of eukaryotic cells through protein complexes known as linkers of the nucleoskeleton and cytoskeleton (LINC complexes). We showed that LINC complexes are highly stable under tensile forces, and that the transmission of force across the complex depends highly on the unique intermolecular covalent bonds formed between the two proteins that construct the complex. Finally, we presented a model for the molecular mechanisms of LINC complex activation and regulation at the nuclear envelope.