Cancer cell migration through and away from tumors is driven in part by migration along aligned extracellular matrix, a process known as contact guidance (CG). To concurrently study the influence of ...architectural and mechanical regulators of CG sensing, we developed a set of CG platforms. Using flat and nanotextured substrates with variable architectures and stiffness, we show that CG sensing is regulated by substrate stiffness and define a mechanical role for microtubules and actomyosin-microtubule interactions during CG sensing. Furthermore, we show that Arp2/3-dependent lamellipodia dynamics can compete with aligned protrusions to diminish the CG response and define Arp2/3- and Formins-dependent actin architectures that regulate microtubule-dependent protrusions, which promote the CG response. Thus, our work represents a comprehensive examination of the physical mechanisms influencing CG sensing.
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•Lamellipodia and microtubule dynamics regulate contact guidance from flat nanolines•The microtubule network acts as an intracellular mechano-structural scaffold•Microtubule scaffolds regulate steric interactions with nanotopographic guidance cues•Arp2/3 and Formins-dependent actin architectures compete to regulate contact guidance
Aligned extracellular matrix architectures in tumors direct migration of invasive cancer cells. Tabdanov et al. show that the mechanical properties of aligned extracellular matrix environments influence invasive cell behavior and define a mechanical role for microtubules and actomyosin-microtubule interactions during sensing of contact guidance cues that arise from aligned extracellular matrix.
Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related ...therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.
Contact guidance due to extracellular matrix architecture is a key regulator of carcinoma invasion and metastasis, yet our understanding of how cells sense guidance cues is limited. Here, using a ...platform with variable stiffness that facilitates uniaxial or biaxial matrix cues, or competing E-cadherin adhesions, we demonstrate distinct mechanoresponsive behavior. Through disruption of traction forces, we observe a profound phenotypic shift towards a mode of dendritic protrusion and identify bimodal processes that govern guidance sensing. In contractile cells, guidance sensing is strongly dependent on formins and FAK signaling and can be perturbed by disrupting microtubule dynamics, while low traction conditions initiate fluidic-like dendritic protrusions that are dependent on Arp2/3. Concomitant disruption of these bimodal mechanisms completely abrogates the contact guidance response. Thus, guidance sensing in carcinoma cells depends on both environment architecture and mechanical properties and targeting the bimodal responses may provide a rational strategy for disrupting metastatic behavior.
Advances in protein design have brought us within reach of developing a nanoscale programming language, in which molecules serve as operands and their conformational states function as logic gates ...with precise input and output behaviors. Combining these nanoscale computing agents into larger molecules and molecular complexes will allow us to write and execute "code". Here, in an important step toward this goal, we report an engineered, single protein design that is allosterically regulated to function as a 'two-input logic OR gate'. Our system is based on chemo- and optogenetic regulation of focal adhesion kinase. In the engineered FAK, all of FAK domain architecture is retained and key intramolecular interactions between the kinase and the FERM domains are externally controlled through a rapamycin-inducible uniRapR module in the kinase domain and a light-inducible LOV2 module in the FERM domain. Orthogonal regulation of protein function was possible using the chemo- and optogenetic switches. We demonstrate that dynamic FAK activation profoundly increased cell multiaxial complexity in the fibrous extracellular matrix microenvironment and decreased cell motility. This work provides proof-of-principle for fine multimodal control of protein function and paves the way for construction of complex nanoscale computing agents.
Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of ...mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.
We enhance activation of primary human T cells by increasing sensitivity of TCR complex to the mechanical signals provided by biomimetic antigen. We achieve this effect by using nucleic acid nanoassemblies for tethering antigen to the presenting surface. Our results highlight the important role for nanoscale configuration of mechanical forces within the TCR-pMHC complex during antigen presentation. Display omitted
Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In ...this study, we report an antagonistic mechanical balance within the dynein–kinesin microtubular motor system. Dynein activity drives the microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion, increasing the topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubule translocation of septin-9 enhances kinesin–MT interactions, outbalances the activity of kinesins over that of dyneins, and induces the discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell–microenvironment interactions.
The principal cause of death in cancer patients is metastasis, which remains an unresolved problem. Conventionally, metastatic dissemination is linked to actomyosin-driven cell locomotion. However, ...the locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, a complementary mechanism of metastatic locomotion powered by dynein-generated forces is identified. These forces arise within a non-stretchable microtubule network and drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. It is also shown that the dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network formed by spatially confining granular hydrogel scaffolds (GHS) made up of microscale hydrogel particles (microgels). These results indicate that the complementary motricity mediated by dynein is always necessary and, in certain instances, sufficient for disseminating metastatic breast cancer cells. These findings advance the fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.
Contact guidance refers to the ability of cells to sense the geometrical features of the microenvironment and respond by changing their shape and adopting the appropriate orientation. Inhibition and ...ablation of nonmuscle myosin 2 (NM2) paralogues have demonstrated their importance for contact guidance. However, the specific roles of the NM2 paralogues have not been systematically studied. In this work we use micropatterned substrates to examine the roles of NM2A and NM2B and to elucidate the relationship of the microenvironment, actomyosin, and microtubules in contact guidance. We show that contact guidance is preserved following loss of NM2B and that expression of NM2A alone is sufficient to establish an appropriate orientation of the cells. Loss of NM2B and overexpression of NM2A result in a prominent cell polarization that is found to be linked to the increased alignment of microtubules with the actomyosin scaffold. Suppression of actomyosin with blebbistatin reduces cell polarity on a flat surface, but not on a surface with contact guidance cues. This indicates that the lost microtubule-actomyosin interactions are compensated for by microtubule-microenvironment interactions, which are sufficient to establish cell polarity through contact guidance.
Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that ...overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize.
Single-protein-based devices that integrate signal sensing with logical operations to generate functional outputs offer exceptional promise for monitoring and modulating biological systems. ...Engineering such intelligent nanoscale computing agents is challenging, as it requires the integration of sensor domains into a functional protein via intricate allosteric networks. We incorporate a rapamycin-sensitive sensor (uniRapR) and a blue light-responsive LOV2 domain into human Src kinase, creating a protein device that functions as a noncommutative combinatorial logic circuit. In our design, rapamycin activates Src kinase, causing protein localization to focal adhesions, whereas blue light exerts the reverse effect that inactivates Src translocation. Focal adhesion maturation induced by Src activation reduces cell migration dynamics and shifts cell orientation to align along collagen nanolane fibers. Using this protein device, we reversibly control cell orientation by applying the appropriate input signals, a framework that may be useful in tissue engineering and regenerative medicine.
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