Mutual antagonism between CD44 and integrins in glioblastoma cell traction and migration.

Cell migration is the major driver of invasion and metastasis during cancer progression. For cells to migrate, they utilize the actin-myosin cytoskeleton and adhesion molecules, such as integrins and CD44, to generate traction forces in their environment. CD44 primarily binds to hyaluronic acid (HA) and integrins primarily bind to extracellular matrix (ECM) proteins such as collagen. However, the role of CD44 under integrin-mediated conditions and vice versa is not well known. Here, we performed traction force microscopy (TFM) on U251 cells seeded on collagen I-coated polyacrylamide gels to assess the functional mechanical relationship between integrins and CD44. Performing TFM on integrin-mediated adhesion conditions, i.e., collagen, we found that CD44KO U251 cells exerted more traction force than wild-type (WT) U251 cells. Furthermore, untreated WT and CD44-blocked WT exhibited comparable results. Conversely, in CD44-mediated adhesive conditions, integrin-blocked WT cells exerted a higher traction force than untreated WT cells. Our data suggest that CD44 and integrins have a mutually antagonistic relationship where one receptor represses the other's ability to generate traction force on its cognate substrate.

"Forcing" new interpretations of molecular tension sensor studies.

Molecular tension sensors are central tools for mechanobiology studies but have limitations in interpretation. Reporting in Cell Reports Methods, Shoyer et al. discover that fluorescent protein photoswitching in concert with sensor extension may expand the use and interpretation of common force-sensing tools.

HUHgle: An Interactive Substrate Design Tool for Covalent Protein-ssDNA Labeling Using HUH-Tags.

HUH-tags have emerged as versatile fusion partners that mediate sequence specific protein-ssDNA bioconjugation through a simple and efficient reaction. Here we present HUHgle, a python-based interactive tool for the visualization, design, and optimization of substrates for HUH-tag mediated covalent labeling of proteins of interest with ssDNA substrates of interest. HUHgle streamlines design processes by integrating an intuitive plotting interface with a search function capable of predicting and displaying protein-ssDNA bioconjugate formation efficiency and specificity in proposed HUH-tag/ssDNA sequence combinations. Validation demonstrates that HUHgle accurately predicts product formation of HUH-tag mediated bioconjugation for single- and orthogonal-labeling reactions. In order to maximize the accessibility and utility of HUHgle, we have implemented it as a user-friendly Google Colab notebook which facilitates broad use of this tool, regardless of coding expertise.

The crystal structure of the human smacovirus 1 Rep domain.

Replication initiator proteins (Reps) from the HUH endonuclease family process specific single-stranded DNA sequences to initiate rolling-circle replication in viruses. Here, the first crystal structure of the apo state of a Rep domain from the smacovirus family is reported. The structure of the human smacovirus 1 Rep domain was obtained at 1.33 Šresolution and represents an expansion of the HUH endonuclease superfamily, allowing greater diversity in bioconjugation-tag applications.

RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes.

Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis.

Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin.

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.

Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH Endonucleases.

Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the -4 and +1 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair's identity (i.e., A·T, T·A, G·C, or C·G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical -4T·+1A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps. IMPORTANCE Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses are a ubiquitous group of viruses that infect organisms across all domains of life. These viruses negatively impact both agriculture and human health. All members of this viral family employ a multifunctional nuclease (Rep) to initiate replication. Reps are structurally similar throughout this family, making them targets of interest for viral inhibition strategies. Here, we investigate the functional dependencies of the Rep protein from Muscovy duck circovirus for ssDNA interaction. We demonstrate that this Rep requires an intramolecular Watson-Crick base pairing for origin of replication (Ori) recognition and interaction. We show that noncognate base pair swaps are well tolerated, highlighting a local structural specificity over sequence specificity. Bioinformatic analysis found that the vast majority of CRESS-DNA Oris form base pairs in conserved positions, suggesting this pairing is a universal requirement for replication initiation in the CRESS-DNA virus family.

Dystrophin missense mutations alter focal adhesion tension and mechanotransduction.

Dystrophin is an essential muscle protein that contributes to cell membrane stability by mechanically linking the actin cytoskeleton to the extracellular matrix via an adhesion complex called the dystrophin-glycoprotein complex. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions (FAs) are also mechanosensitive adhesion complexes that connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and FA force transmission has not been investigated. Using a vinculin-based bioluminescent tension sensor, we measured FA tension in transgenic C2C12 myoblasts expressing wild-type (WT) dystrophin, a nonpathogenic single nucleotide polymorphism (SNP) (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). Our data revealed cross talk between dystrophin and FAs, as the expression of WT or I232M dystrophin increased FA tension compared to dystrophin-less nontransgenic myoblasts. In contrast, the expression of L54R or L172H did not increase FA tension, indicating that these disease-causing mutations compromise the mechanical function of dystrophin as an FA allosteric regulator. Decreased FA tension caused by these mutations manifests as defective migration, as well as decreased Yes-associated protein 1 (YAP) activation, possibly by the disruption of the ability of FAs to transmit forces between the extracellular matrix and cytoskeleton. Our results indicate that dystrophin influences FA tension and suggest that dystrophin disease-causing missense mutations may disrupt a cellular tension-sensing pathway in dystrophic skeletal muscle.

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity.

Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.

Enhanced Molecular Tension Sensor Based on Bioluminescence Resonance Energy Transfer (BRET).

Molecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically encoded tension sensors use FRET to report on extension of a deformable peptide encoded in a cellular protein of interest. Here, we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET), which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET tension sensor will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.

Programmable Assembly of Adeno-Associated Virus-Antibody Composites for Receptor-Mediated Gene Delivery.

Adeno-associated virus (AAV) has emerged as a viral gene delivery vector that is safe in humans, able to infect both dividing and arrested cells and drive long-term expression (>6 months). Unfortunately, the naturally evolved properties of many AAV serotypes-including low cell type specificity and largely overlapping tropism-are mismatched to applications that require cell type-specific infection, such as neural circuit mapping or precision gene therapy. A variety of approaches to redirect AAV tropism exist, but there is still the need for a universal solution for directing AAV tropism toward user-defined cellular receptors that does not require extensive case-by-case optimization and works with readily available components. Here, we report AAV engineering approaches that enable programmable receptor-mediated gene delivery. First, we genetically encode small targeting scaffolds into a variable region of an AAV capsid and show that this redirects tropism toward the receptor recognized by these targeting scaffolds and also renders this AAV variant resistant to neutralizing antibodies present in nonhuman primate serum. We then simplify retargeting of tropism by engineering the same variable loop to encode a HUH tag, which forms a covalent bond to single-stranded DNA oligos conjugated to store-bought antibodies. We demonstrate that retargeting this HUH-AAVs toward different receptors is as simple as "arming" a premade noninfective AAV template with a different antibody in a conjugation process that uses widely available reagents and requires no optimization or extensive purification. Composite antibody-AAV nanoparticles structurally separate tropism and payload encapsulation, allowing each to be engineered independently.

Author Correction: Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Crystal structure of the Wheat dwarf virus Rep domain.

The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein-ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein-DNA interactions that are programmable. The structure of the WDV Rep domain reported here was solved in the apo state from a crystal diffracting to 1.24 Šresolution and represents an initial step in the direction of solving the structure of a protein-ssDNA complex.

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.

The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.

A toolkit for studying cell surface shedding of diverse transmembrane receptors.

Proteolysis of transmembrane receptors is a critical cellular communication mechanism dysregulated in disease, yet decoding proteolytic regulation mechanisms of hundreds of shed receptors is hindered by difficulties controlling stimuli and unknown fates of cleavage products. Notch proteolytic regulation is a notable exception, where intercellular forces drive exposure of a cryptic protease site within a juxtamembrane proteolytic switch domain to activate transcriptional programs. We created a Synthetic Notch Assay for Proteolytic Switches (SNAPS) that exploits the modularity and unequivocal input/response of Notch proteolysis to screen surface receptors for other putative proteolytic switches. We identify several new proteolytic switches among receptors with structural homology to Notch. We demonstrate SNAPS can detect shedding in chimeras of diverse cell surface receptors, leading to new, testable hypotheses. Finally, we establish the assay can be used to measure modulation of proteolysis by potential therapeutics and offer new mechanistic insights into how DECMA-1 disrupts cell adhesion.

Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template.

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to a 30-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci and is more pronounced at low RNP concentrations.

The Molecular Mechanism of Notch Activation.

Research in the last several years has shown that Notch proteolysis, and thus Notch activation, is conformationally controlled by the extracellular juxtamembrane NRR of Notch, which sterically occludes the S2 protease site until ligand binds. The question of how conformational exposure of the protease site is achieved during physiologic activation, and thus how normal activation is bypassed in disease pathogenesis, has been the subject of intense study in the last several years, and is the subject of this chapter. Here, we summarize the structural features of the NRR domains of Notch receptors that establish the autoinhibited state and then review a number of recent studies aimed at testing the mechanotransduction model for Notch signaling using force spectroscopy and molecular tension sensors.

Sequence-Directed Covalent Protein-DNA Linkages in a Single Step Using HUH-Tags.

We present a robust strategy to covalently link proteins and DNA using HUH-endonuclease domains as fusion partners (HUH-tags). We show that HUH-tags react robustly with specific sequences of unmodified single-stranded DNA, and we have identified five tags that react orthogonally with distinct DNA sequences. We demonstrate the versatility of HUH-tags as fusion partners in Cas9-mediated gene editing and the construction of doubly DNA-tethered proteins for single-molecule studies. Finally we demonstrate application to cellular imaging in live and fixed cells.

Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch.

Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the Notch1 regulatory switch results in sensitivity to metalloprotease cleavage, and bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction.