Mechanism of exportin retention in the cell nucleus.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinß1 (Kapß1 or importinß1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapß1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

High Throughput Blood-brain Barrier Organoid Generation and Assessment of Receptor-Mediated Antibody Transcytosis.

Targeting receptor-mediated transcytosis (RMT) is a successful strategy for drug delivery of biologic agents across the blood-brain barrier (BBB). The recent development of human BBB organoid models is a major advancement to help characterize the mechanisms of RMT and thus accelerate the design of brain delivery technologies. BBB organoids exhibit self-organization, which resembles the architecture of the neurovascular unit, and low paracellular permeability, due to the formation of tight junctions between endothelial cells. However, current methods of organoid generation have low throughput, exhibit substantial heterogeneity across experiments, and require extensive manual handling. These limitations prevent the use of BBB organoids as a screening tool for discovery and optimization of therapeutic molecules. In this protocol, we use hydrogel-based arrays to generate human BBB organoids, with a 35-fold increase in organoid yield as compared to previous protocols using 96-well plates. We incubate BBB organoid arrays with monoclonal antibody-based constructs and use a custom semi-automated imaging assay to assess RMT within the organoid core. The experimental and analytical tools described in this protocol provide a scalable platform that can be incorporated in the early stages of drug discovery to accelerate the development and optimization of brain delivery technologies to cross the BBB.

Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays.

BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Extracellular Matrix Geometry and Initial Adhesive Position Determine Stress Fiber Network Organization during Cell Spreading.

Three-dimensional matrices often contain highly structured adhesive tracks that require cells to turn corners and bridge non-adhesive areas. Here, we investigate these complex processes using micropatterned cell adhesive frames. Spreading kinetics on these matrices depend strongly on initial adhesive position and are predicted by a cellular Potts model (CPM), which reflects a balance between adhesion and intracellular tension. As cells spread, new stress fibers (SFs) assemble periodically and parallel to the leading edge, with spatial intervals of ∼2.5 µm, temporal intervals of ∼15 min, and characteristic lifetimes of ∼50 min. By incorporating these rules into the CPM, we can successfully predict SF network architecture. Moreover, we observe broadly similar behavior when we culture cells on arrays of discrete collagen fibers. Our findings show that ECM geometry and initial cell position strongly determine cell spreading and that cells encode a memory of their spreading history through SF network organization.

The role of nucleocytoplasmic transport in mechanotransduction.

Cells integrate mechanical and biochemical signals via a process called mechanotransduction to generate essential gene expression patterns in space and time. This is vital for cell migration and proliferation as well as tissue morphogenesis and remodeling. While the force-sensing and force-transducing mechanisms are generally known, it remains unclear how mechanoresponsive transcription factors (TFs) are selectively translocated into the nucleus upon force activation. Such TFs include Yes-Associated Protein (YAP), Myocardin Related Transcription Factors (MRTFs), Hypoxia Induced Factors (HIFs) and others. Here, we discuss how the nucleocytoplasmic transport machinery intersects with mechanoresponsive TFs to facilitate their selective transport through nuclear pore complexes.

Actomyosin stress fiber subtypes have unique viscoelastic properties and roles in tension generation.

Actomyosin stress fibers (SFs) support cell shape and migration by directing intracellular tension to the extracellular matrix (ECM) via focal adhesions. Migrating cells exhibit three SF subtypes (dorsal SFs, transverse arcs, and ventral SFs), which differ in their origin, location, and ECM connectivity. While each subtype is hypothesized to play unique structural roles, this idea has not been directly tested at the single-SF level. Here, we interrogate the mechanical properties of single SFs of each subtype based on their retraction kinetics following laser incision. While each SF subtype bears distinct mechanical properties, these properties are highly interdependent, with incision of dorsal fibers producing centripetal recoil of adjacent transverse arcs and the retraction of incised transverse arcs being limited by attachment points to dorsal SFs. These observations hold whether cells are allowed to spread freely or are confined to crossbow ECM patterns. Consistent with this interdependence, subtype-specific knockdown of dorsal SFs (palladin) or transverse arcs (mDia2) influences ventral SF retraction. These altered mechanics are partially phenocopied in cells cultured on ECM microlines that preclude assembly of dorsal SFs and transverse arcs. Our findings directly demonstrate that different SF subtypes play distinct roles in generating tension and form a mechanically interdependent network.

Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation.

The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.

Emergent cellular self-organization and mechanosensation initiate follicle pattern in the avian skin.

The spacing of hair in mammals and feathers in birds is one of the most apparent morphological features of the skin. This pattern arises when uniform fields of progenitor cells diversify their molecular fate while adopting higher-order structure. Using the nascent skin of the developing chicken embryo as a model system, we find that morphological and molecular symmetries are simultaneously broken by an emergent process of cellular self-organization. The key initiators of heterogeneity are dermal progenitors, which spontaneously aggregate through contractility-driven cellular pulling. Concurrently, this dermal cell aggregation triggers the mechanosensitive activation of ß-catenin in adjacent epidermal cells, initiating the follicle gene expression program. Taken together, this mechanism provides a means of integrating mechanical and molecular perspectives of organ formation.

Geometry and network connectivity govern the mechanics of stress fibers.

Actomyosin stress fibers (SFs) play key roles in driving polarized motility and generating traction forces, yet little is known about how tension borne by an individual SF is governed by SF geometry and its connectivity to other cytoskeletal elements. We now address this question by combining single-cell micropatterning with subcellular laser ablation to probe the mechanics of single, geometrically defined SFs. The retraction length of geometrically isolated SFs after cutting depends strongly on SF length, demonstrating that longer SFs dissipate more energy upon incision. Furthermore, when cell geometry and adhesive spacing are fixed, cell-to-cell heterogeneities in SF dissipated elastic energy can be predicted from varying degrees of physical integration with the surrounding network. We apply genetic, pharmacological, and computational approaches to demonstrate a causal and quantitative relationship between SF connectivity and mechanics for patterned cells and show that similar relationships hold for nonpatterned cells allowed to form cell-cell contacts in monolayer culture. Remarkably, dissipation of a single SF within a monolayer induces cytoskeletal rearrangements in cells long distances away. Finally, stimulation of cell migration leads to characteristic changes in network connectivity that promote SF bundling at the cell rear. Our findings demonstrate that SFs influence and are influenced by the networks in which they reside. Such higher order network interactions contribute in unexpected ways to cell mechanics and motility.

N-terminal specific conjugation of extracellular matrix proteins to 2-pyridinecarboxaldehyde functionalized polyacrylamide hydrogels.

Polyacrylamide hydrogels have been used extensively to study cell responses to the mechanical and biochemical properties of extracellular matrix substrates. A key step in fabricating these substrates is the conjugation of cell adhesion proteins to the polyacrylamide surfaces, which typically involves nonspecifically anchoring these proteins via side-chain functional groups. This can result in a loss of presentation control and altered bioactivity. Here, we describe a new functionalization strategy in which we anchor full-length extracellular matrix proteins to polyacrylamide substrates using 2-pyridinecarboxaldehyde, which can be co-polymerized into polyacrylamide gels and used to immobilize proteins by their N-termini. This one-step reaction proceeds under mild aqueous conditions and does not require additional reagents. We demonstrate that these substrates can readily conjugate to various extracellular matrix proteins, as well as promote cell adhesion and spreading. Notably, this chemistry supports the assembly and cellular remodeling of large collagen fibers, which is not observed using conventional side-chain amine-conjugation chemistry.

A biomechanical perspective on stress fiber structure and function.

Stress fibers are actomyosin-based bundles whose structural and contractile properties underlie numerous cellular processes including adhesion, motility and mechanosensing. Recent advances in high-resolution live-cell imaging and single-cell force measurement have dramatically sharpened our understanding of the assembly, connectivity, and evolution of various specialized stress fiber subpopulations. This in turn has motivated interest in understanding how individual stress fibers generate tension and support cellular structure and force generation. In this review, we discuss approaches for measuring the mechanical properties of single stress fibers. We begin by discussing studies conducted in cell-free settings, including strategies based on isolation of intact stress fibers and reconstitution of stress fiber-like structures from purified components. We then discuss measurements obtained in living cells based both on inference of stress fiber properties from whole-cell mechanical measurements (e.g., atomic force microscopy) and on direct interrogation of single stress fibers (e.g., subcellular laser nanosurgery). We conclude by reviewing various mathematical models of stress fiber function that have been developed based on these experimental measurements. An important future challenge in this area will be the integration of these sophisticated biophysical measurements with the field's increasingly detailed molecular understanding of stress fiber assembly, dynamics, and signal transduction. This article is part of a Special Issue entitled: Mechanobiology.