Hengameh Shams

Mechanotransduction pathways linking the extracellular matrix to the nucleus.

Cells contain several mechanosensing components that transduce mechanical signals into biochemical cascades. During cell-ECM adhesion, a complex network of molecules mechanically couples the extracellular matrix (ECM), cytoskeleton, and nucleoskeleton. The network comprises transmembrane receptor proteins and focal adhesions, which link the ECM and cytoskeleton. Additionally, recently identified protein complexes extend this linkage to the nucleus by linking the cytoskeleton and the nucleoskeleton. Despite numerous studies in this field, due to the complexity of this network, our knowledge of the mechanisms of cell-ECM adhesion at the molecular level remains remarkably incomplete. Herein, we present a review of the structures of key molecules involved in cell-ECM adhesion, along with an evaluation of their predicted roles in mechanical sensing. Additionally, specific binding events prompted by force-induced conformational changes of each molecule are discussed. Finally, we propose a model for the biomechanical events prominent in cell-ECM adhesion.

Actin Reorganization through Dynamic Interactions with Single-Wall Carbon Nanotubes

Single-wall carbon nanotubes (SWCNTs) have been widely used for biological applications in recent years, and thus, it is critical to understand how these inert nanomaterials influence cell behavior. Recently, it has been observed that cellular phenotypes such as proliferation, force generation and growth change upon SWCNT treatment, and SWCNTs directly affect the organization and redistribution of the actin cytoskeleton. However, the interactions between SWCNTs and actin at the molecular level or how this interaction changes actin structure remain largely unknown. Here, we investigated direct interaction of actin with SWCNT using all-atom molecular dynamics simulations and NIR spectroscopy of actin-dispersed SWCNTs. Actin can stably bind to the SWCNT surfaces via hydrophobic interactions but still allows nanotubes to slide and rotate on the actin surface. Our results establish several nanoscale conformational changes for the actin-SWCNT complexes, and we suggest these changes likely induce reorganization of actin filaments observed at larger scales.

A Molecular Trajectory of α-Actinin Activation

The mechanisms by which living cells respond to mechanical stimuli are not yet fully understood. It has been suggested that mechanosensing proteins play an important role in mechanotransduction because their binding affinities are directly affected by the external stress. α-Actinin is an actin cross-linker and may act as a mechanosensor in adhesion sites. Its interaction with vinculin is suggested to be mechanically regulated. In this study, the free energy of activation is explored using the umbrella sampling method. An activation trajectory is generated in which α-actinins vinculin-binding site swings out of the rod domain, leading to approximately an 8 kcal/mol free energy release. The activation trajectory reveals several local and global conformational changes along the activation pathway accompanied by the breakage of a number of key interactions stabilizing the inhibited structure. These results may shed light on the role of α-actinin in cellular mechanotransduction and focal adhesion formation.

Altered cell mechanics from the inside: dispersed single wall carbon nanotubes integrate with and restructure actin.

With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs) are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

A Disulfide Bond Is Required for the Transmission of Forces through SUN-KASH Complexes

Numerous biological functions of a cell, including polarization, differentiation, division, and migration, rely on its ability to endure mechanical forces generated by the cytoskeleton on the nucleus. Coupling of the cytoskeleton and nucleoskeleton is ultimately mediated by LINC complexes that are formed via a strong interaction between SUN- and KASH-domain-containing proteins in the nuclear envelope. These complexes are mechanosensitive and essential for the transmission of forces between the cytoskeleton and nucleoskeleton, and the progression of cellular mechanotransduction. Herein, using molecular dynamics, we examine the effect of tension on the human SUN2-KASH2 complex and show that it is remarkably stable under physiologically relevant tensile forces and large strains. However, a covalent disulfide bond between two highly conserved cysteine residues of SUN2 and KASH2 is crucial for the stability of this interaction and the transmission of forces through the complex.

Enhanced intracellular delivery of small molecules and drugs via non-covalent ternary dispersions of single-wall carbon nanotubes

Albumins are used biologically and pharmacologically as transport proteins to deliver molecules to cells. Albumins also efficiently coat single-wall carbon nanotubes (SWCNTs) and promote their entry into mammalian and immune cells by the millions. Here, we show SWCNTs dispersed with bovine serum albumin (BSA) that are pre-loaded with rhodamine B (RB), small hydrophobic dye molecules that we consider here as models for drugs, drastically increase delivery of RB to HeLa cells and macrophages in culture. We determine spatial and concentration distribution of RB by independently visualizing SWCNTs and RB within the cells using unique SWCNT NIR fluorescence and fluorescence lifetime imaging of RB. The SWCNTs-BSA-RB ternary complexes are stable in water for days, and RB is only released when BSA is thermally or enzymatically denatured. We demonstrate efficacy of this approach by delivering daunomycin, a fluorescent chemotherapeutic drug that reduces proliferation in HeLa cells. Furthermore, we use molecular dynamics simulations to identify separate regions in BSA for drug loading and binding to SWCNTs. Together, our results demonstrate a pathway to enhance the delivery of a wide variety of drugs to cells through SWCNTs coated with albumin pre-loaded with drug molecules.

Dynamic Regulation of α-Actinin’s Calponin Homology Domains on F-Actin

α-Actinin is an essential actin cross-linker involved in cytoskeletal organization and dynamics. The molecular conformation of α-actinins actin-binding domain (ABD) regulates its association with actin and thus mutations in this domain can lead to severe pathogenic conditions. A point mutation at lysine 255 in human α-actinin-4 to glutamate increases the binding affinity resulting in stiffer cytoskeletal structures. The role of different ABD conformations and the effect of K255E mutation on ABD conformations remain elusive. To evaluate the impact of K255E mutation on ABD binding to actin we use all-atom molecular dynamics and free energy calculation methods and study the molecular mechanism of actin association in both wild-type α-actinin and in the K225E mutant. Our models illustrate that the strength of actin association is indeed sensitive to the ABD conformation, predict the effect of K255E mutation--based on simulations with the K237E mutant chicken α-actinin--and evaluate the mechanism of α-actinin binding to actin. Furthermore, our simulations showed that the calmodulin domain binding to the linker region was important for regulating the distance between actin and ABD. Our results provide valuable insights into the molecular details of this critical cellular phenomenon and further contribute to an understanding of cytoskeletal dynamics in health and disease.

The ?stressful? life of adhesion molecules: On the molecular mechanosensitivity of adhesomes

Cells have evolved into complex sensory machines that communicate with their microenvironment via mechanochemical signaling. Extracellular mechanical cues trigger complex biochemical pathways in the cell, which regulate various cellular processes. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes, also known as the integrin adhesome, that link the extracellular matrix (ECM) to the actin cytoskeleton, and are part of powerful intracellular machinery orchestrating mechanotransduction pathways. As forces are transmitted across FAs, individual proteins undergo structural and functional changes that involve a conversion of chemical to mechanical energy. The local composition of early adhesions likely defines the regional stress levels and determines the type of newly recruited proteins, which in turn modify the local stress distribution. Various approaches have been used for detecting and exploring molecular mechanisms through which FAs are spatiotemporally regulated, however, many aspects are yet to be understood. Current knowledge on the molecular mechanisms of mechanosensitivity in adhesion proteins is discussed herein along with important questions yet to be addressed, are discussed.

α-Actinin Induces a Kink in the Transmembrane Domain of β3-Integrin and Impairs Activation via Talin

Integrin-mediated signaling is crucial for cell-substrate adhesion and can be triggered from both intra- and extracellular interactions. Although talin binding is sufficient for inside-out activation of integrin, other cytoplasmic proteins such as α-actinin and filamin can directly interfere with talin-mediated integrin activation. Specifically, α-actinin plays distinct roles in regulating αIIbβ3 versus α5β1 integrin. It has been shown that α-actinin competes with talin for binding to the cytoplasmic tail of β3-integrin, whereas it cooperates with talin for activating integrin α5β1. In this study, molecular dynamics simulations were employed to compare and contrast molecular mechanisms of αIIbβ3 and α5β1 activation in the presence and absence of α-actinin. Our results suggest that α-actinin impairs integrin signaling by both undermining talin binding to the β3-integrin cytoplasmic tail and inducing a kink in the transmembrane domain of β3-integrin. Furthermore, we showed that α-actinin promote talin association with β1-integrin by restricting the motion of the cytoplasmic tail and reducing the entropic barrier for talin binding. Taken together, our results showed that the interplay between talin and α-actinin regulates signal transmission via controlling the conformation of the transmembrane domain and altering natural response modes of integrins in a type-specific manner.