Engineered adhesion molecules drive synapse organization.

In multicellular organisms, cell-adhesion molecules connect cells into tissues and mediate intercellular signaling between these cells. In vertebrate brains, synaptic cell-adhesion molecules (SAMs) guide the formation, specification, and plasticity of synapses. Some SAMs, when overexpressed in cultured neurons or in heterologous cells co-cultured with neurons, drive formation of synaptic specializations onto the overexpressing cells. However, genetic deletion of the same SAMs from neurons often has no effect on synapse numbers, but frequently severely impairs synaptic transmission, suggesting that most SAMs control the function and plasticity of synapses (i.e., organize synapses) instead of driving their initial establishment (i.e., make synapses). Since few SAMs were identified that mediate initial synapse formation, it is difficult to develop methods that enable experimental control of synaptic connections by targeted expression of these SAMs. To overcome this difficulty, we engineered novel SAMs from bacterial proteins with no eukaryotic homologues that drive synapse formation. We named these engineered adhesion proteins "Barnoligin" and "Starexin" because they were assembled from parts of Barnase and Neuroligin-1 or of Barstar and Neurexin3ß, respectively. Barnoligin and Starexin robustly induce the formation of synaptic specializations in a specific and directional manner in cultured neurons. Synapse formation by Barnoligin and Starexin requires both their extracellular Barnase- and Barstar-derived interaction domains and their Neuroligin- and Neurexin-derived intracellular signaling domains. Our findings support a model of synapse formation whereby trans-synaptic interactions by SAMs drive synapse organization via adhesive interactions that activate signaling cascades.

A Bioengineering Strategy to Control ADAM10 Activity in Living Cells.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer's disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-ß precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.

Biosynthetic Structural Proteins with Super Plasticity, Extraordinary Mechanical Performance, Biodegradability, Biocompatibility and Information Storage Ability.

Degradable bioplastics have attracted growing interest worldwide. However, it is challenging to develop bioplastics with a simple processing procedure, strong mechanical performance, good biocompatibility, and adjustable physicochemical properties. Herein, we introduced structural proteins as building blocks and developed a simple environmentally friendly approach to fabricate diverse protein-based plastics. A cost-effective and high-level production approach was developed through batch fermentation of Escherichia coli to produce the biomaterials. These bioplastics possess super plasticity, biocompatibility, biodegradability, and high resistance to organic solvents. Their structural and mechanical properties can be precisely controlled. Besides, high density information storage and hemostatic applications were realized in the bioplastic system. The customizable bioplastics have great potential for applications in numerous fields and are capable to scale up to the industrial level.

Introducing an artificial photo-switch into a biological pore: A model study of an engineered α-hemolysin.

In recent years, engineered biological pores responsive to external stimuli have been fruitfully used for various biotechnological applications. Moreover, the strategy of tethering photo-switchable moieties into biomolecules has provided an unprecedented temporal control of purposely designed nanodevices, as demonstrated, for example, by the light-mediated regulation of the activity of enzymes and biochannels. Inspired by these advancements, we propose here a de novo designed nanodevice featuring the α-hemolysin (αHL) membrane channel purposely functionalized by an artificial "on/off" molecular switch. The switch, which is based on the photo-isomerization of the azobenzene moiety, introduces a smart nano-valve into the natural non-gated pore to confer tunable transport properties. We validated through molecular dynamics simulations and free energy calculations the effective inter-conversion of the engineered αHL pore between two configurations corresponding to an "open" and a "closed" form. The reported switchable translocation of a single-stranded DNA fragment under applied voltage supports the promising capabilities of this nanopore prototype in view of molecular sensing, detection and delivery applications at single-molecule level.

Amoeba-inspired magnetic microgel assembly assisted by engineered dextran-binding protein for vaccination against life-threatening systemic infection.

Vaccination is critical for population protection from pathogenic infections. However, its efficiency is frequently compromised by a failure of antigen retention and presentation. Herein, we designed a dextran-binding protein DexBP, which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A, together with the sequence of the fluorescent protein mCherry. DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles. The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol) framework and a reactive oxygen species-responsive linker, obtaining magnetic polymeric microgels for carrying pathogen antigen. Similar to amoeba aggregation, the microgels self-assembled to form aggregates and further induced dendritic cell aggregation. This step-by-step assembly retained antigens at lymph nodes, promoted antigen presentation, stimulated humoral immunity, and protected the mice from life-threatening systemic infections. This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections. Electronic Supplementary Material: Supplementary material (AFM image and zeta potential of MG; TEM, FT-IR, DLS, and zeta potential of MNP-DexBP; zeta potential of MG+CaAg and MG+MNP-DexBP+CaAg; antigen release profile of MG+CaAg and MG+MNP-DexBP+CaAg; aggregation and dispersion of dendritic cells induced by MG+MNP-DexBP+CaAg; uptake of FITC-labeled CaAg (fCaAg) and intracellular distribution of fCaAg in the dendritic cells; antigen retention and dendritic cell activation in lymph nodes; and serum anti-CaAg antibody levels on day 3 after C. albicans infection in the mice pre-immunized by PBS (control), CaAg, MG+CaAg, and MG+MNP-DexBP+CaAg) is available in the online version of this article at 10.1007/s12274-022-4809-1.

Bionic Engineered Protein Coating Boosting Anti-Biofouling in Complex Biological Fluids.

Implantable medical devices have been widely applied in diagnostics, therapeutics, organ restoration, and other biomedical areas, but often suffer from dysfunction and infections due to irreversible biofouling. Inspired by the self-defensive "vine-thorn" structure of climbing thorny plants, a zwitterion-conjugated protein is engineered via grafting sulfobetaine methacrylate (SBMA) segments on native bovine serum albumin (BSA) protein molecules for surface coating and antifouling applications in complex biological fluids. Unlike traditional synthetic polymers of which the coating operation requires arduous surface pretreatments, the engineered protein BSA@PSBMA (PolySBMA conjugated BSA) can achieve facile and surface-independent coating on various substrates through a simple dipping/spraying method. Interfacial molecular force measurements and adsorption tests demonstrate that the substrate-foulant attraction is significantly suppressed due to strong interfacial hydration and steric repulsion of the bionic structure of BSA@PSBMA, enabling coating surfaces to exhibit superior resistance to biofouling for a broad spectrum of species including proteins, metabolites, cells, and biofluids under various biological conditions. This work provides an innovative paradigm of using native proteins to generate engineered proteins with extraordinary antifouling capability and desired surface properties for bioengineering applications.

Engineered protein and Jakinib nanoplatform with extraordinary rheumatoid arthritis treatment.

Rheumatoid arthritis (RA) is a relatively common inflammatory disease that affects the synovial tissue, eventually results in joints destruction and even long-term disability. Although Janus kinase inhibitors (Jakinibs) show a rapid efficacy and are becoming the most successful agents in RA therapy, high dosing at frequent interval and severe toxicities cannot be avoided. Here, we developed a new type of fully compatible nanocarriers based on recombinant chimeric proteins with outstanding controlled release of upadacitinib. In addition, the fluorescent protein component of the nanocarriers enabled noninvasive fluorescence imaging of RA lesions, thus allowing real-time detection of RA therapy. Using rat models, the nanotherapeutic is shown to be superior to free upadacitinib, as indicated by extended circulation time and sustained bioefficacy. Strikingly, this nanosystem possesses an ultralong half-life of 45 h and a bioavailability of 4-times higher than pristine upadacitinib, thus extending the dosing interval from one day to 2 weeks. Side effects such as over-immunosuppression and leukocyte levels reduction were significantly mitigated. This smart strategy boosts efficacy, safety and visuality of Jakinibs in RA therapy, and potently enables customized designs of nanoplatforms for other therapeutics. Electronic Supplementary Material: Supplementary material (further details of DLS analysis, biocompatibility of PCP-UPA, CIA models construction, etc.) is available in the online version of this article at 10.1007/s12274-023-5838-0.

Bioinspired engineered proteins enable universal anchoring strategy for surface functionalization.

HYPOTHESIS: Conventional coating strategies and materials for bio-applications with protective, diagnostic, and therapeutic functions are commonly limited by their arduous preparation processes and lack of on-demand functionalities. Herein, inspired by the 'root-leaf' structure of grass, a series of novel polyacrylate-conjugated proteins can be engineered with sticky bovine serum albumin (BSA) protein as a 'root' anchoring layer and a multifunctional polyacrylate as a 'leaf' functional layer for the facile coating procedure and versatile surface functionalities. EXPERIMENTS: The engineered proteins were synthesized based on click chemistry, where the 'root' layer can universally anchor onto both organic and inorganic substrates through a facile dip/spraying method with excellent stability in harsh solution conditions, thanks to its multiple adaptive molecular interactions with substrates that further elucidated by molecular force measurements between the 'root' BSA protein and substrates. The 'leaf' conjugated-polyacrylates imparted coatings with versatile on-demand functionalities, such as resistance to over 99% biofouling in complex biofluids, pH-responsive performance, and robust adhesion with various nanomaterials. FINDINGS: By synergistically leveraging the universal anchoring capabilities of BSA with the versatile physicochemical properties of polyacrylates, this study introduces a promising and facile strategy for imparting novel functionalities to a myriad of surfaces through engineering natural proteins and biomaterials for biotechnical and nanotechnical applications.

Building biomaterials through genetic code expansion.

Genetic code expansion (GCE) enables directed incorporation of noncoded amino acids (NCAAs) and unnatural amino acids (UNAAs) into the active core that confers dedicated structure and function to engineered proteins. Many protein biomaterials are tandem repeats that intrinsically include NCAAs generated through post-translational modifications (PTMs) to execute assigned functions. Conventional genetic engineering approaches using prokaryotic systems have limited ability to biosynthesize functionally active biomaterials with NCAAs/UNAAs. Codon suppression and reassignment introduce NCAAs/UNAAs globally, allowing engineered proteins to be redesigned to mimic natural matrix-cell interactions for tissue engineering. Expanding the genetic code enables the engineering of biomaterials with catechols - growth factor mimetics that modulate cell-matrix interactions - thereby facilitating tissue-specific expression of genes and proteins. This method of protein engineering shows promise in achieving tissue-informed, tissue-compliant tunable biomaterials.

Engineered Synthetic STxB for Enhanced Cytosolic Delivery.

Many molecular targets for cancer therapy are located in the cytosol. Therapeutic macromolecules are generally not able to spontaneously translocate across membranes to reach these cytosolic targets. Therefore a strong need exists for tools that enhance cytosolic delivery. Shiga toxin B-subunit (STxB) is used to deliver therapeutic principles to disease-relevant cells that express its receptor, the glycolipid Gb3. Based on its naturally existing membrane translocation capacity, STxB delivers antigens to the cytosol of Gb3-positive dendritic cells, leading to the induction of CD8+ T cells. Here, we have explored the possibility of further increasing the membrane translocation of STxB to enable other therapeutic applications. For this, our capacity to synthesize STxB chemically was exploited to introduce unnatural amino acids at different positions of the protein. These were then functionalized with hydrophobic entities to locally destabilize endosomal membranes. Intracellular trafficking of these functionalized STxB was measured by confocal microscopy and their cytosolic arrival with a recently developed highly robust, sensitive, and quantitative translocation assay. From different types of hydrophobic moieties that were linked to STxB, the most efficient configuration was determined. STxB translocation was increased by a factor of 2.5, paving the path for new biomedical opportunities.

Mannosylated engineered trichosanthin-legumain protein vaccine hydrogel for breast cancer immunotherapy.

The development of cancer vaccines based on tumor-associated antigens is hurdled by lack of an efficient adjuvant and insufficient efficacy. To improve the efficacy of vaccines, a genetically-engineered method was employed in this work to achieve the codelivery of antigen and adjuvant to enhance immune responses. Trichosanthin is a plant-derived protein that possesses cancer immune stimulation function. A genetically engineered protein vaccine composed of trichosanthin (adjuvant) and legumain domain (a peptidic antigen) was constructed, which was further chemically modified with mannose for targeting dendritic cells (DCs). The method is facile and ready for scaling up for massive production. Such a "two-in-one" vaccine is advantageous for codelivery for augmenting the immune responses. The vaccine inhibited the tumors by triggering a robust cytotoxic T lymphocyte response in the orthotopic-breast-tumor mice. Furthermore, the vaccine was loaded into the temperature-sensitive hydrogel based on Pluronic F127 for implanting use in the post-surgical site. The sustained-released vaccine from the hydrogel inhibited not only the tumor recurrence but also the lung metastases of breast cancer. These findings demonstrated that it was a safe and effective vaccination for breast cancer immunotherapy in a prophylactical and therapeutical manner for remodeling the tumor immune microenvironment and arresting tumor growth.

High-Efficiency Treatment for Osteoarthritis via Self-Assembled Dual-Functionalized Nanobiologics.

Osteoarthritis (OA) is a progressive joint disease that has a complex pathogenesis and lacks effective drugs. OA develops with cartilage degeneration and inflammation, thus synthesizing a drug with both anti-inflammatory properties and cartilage-repair capacity provides a promising treatment strategy. Therefore, in this study, we report self-assembled nanobiologics composed of an engineered recombinant IL-1 receptor antagonist (IL-1Ra) chimeric protein with chondroitin sulfate (CS). The nanobiologics, termed ICN, exhibit extraordinary biocompatibility, low immunogenicity, and good bioefficacy. Furthermore, our study revealed that ICN significantly reduced cartilage degradation, inhibited synovial inflammation, and suppressed osteophyte formation in OA rat models. The excellent therapeutic effects on OA can be attributed to the synergistic anti-inflammatory and cartilage-repair properties of ICN's constituents. Thus, our novel strategy offers insights into the development of drugs for OA treatment and research on nanobiomedicine, which can also be adapted for other diseases with similar pathologies.

An Engineered Protein Adhesive with Properties of Tissue Integration and Controlled Release for Efficient Cartilage Repair.

Cartilage damage is a prevalent health concern among humans. The inertness of cartilage, the absence of self-healing properties, and the lack of appropriate repair materials that integrate into the tissue pose a significant challenge for cartilage repair. Thus, it is important to develop novel soft biomaterials with strong tissue adhesion and chondrogenic capabilities for cartilage repair. Herein, a new type of protein adhesive is reported that exhibits superior cartilage repair performance. The material is fabricated by the electrostatic combination of chondroitin sulfate (CS) and positively charged elastin-like protein, which is derived from natural components of the extracellular matrix (ECM). The adhesive showed robust adhesion properties on different tissue substrates, offering a favorable environment for cartilage tissue integration. Noncovalent bonding between CS molecules in the glue allows for its controlled release, which is required for efficient chondrogenic differentiation. When implanted into a rat model of cartilage defect, this protein adhesive exhibited beneficial healing effects, as evidenced by enhanced chondrogenesis, sufficient ECM production, and lateral integration. Therefore, this engineered protein complex is a promising candidate for translational application in the field of cartilage repair.

Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides.

Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (Tt). To do so, this Method paper describes a simple live-cell imaging technique to estimate the Tt of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different Tt, the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower Tt. In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular Tt of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.

Caveolin elastin-like polypeptide fusions mediate temperature-dependent assembly of caveolar microdomains.

Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate caveolin-1 (CAV1). By attaching different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes Cholera Toxin Subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin 1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing.

Gap closure of different shape wounds: In vitro and in vivo experimental models in the presence of engineered protein adhesive hydrogel.

The present study emphasizes the role of engineered protein (gallic acid engineered gelatin [GEG]) on the closure of wound gaps of different shapes assessed under in vitro (fibroblast cell line) and in vivo (rat) experimental models. Circular, triangle, rectangle, and square are the shapes selected for the study. Intending engineered protein (GEG) augments the cell migration in rectangle and triangle shapes and reduces the gap space significantly compared with circular and square shapes. Similar observations were made with in vivo model study, and it was observed that the wound closure starts along the wound edges. In circular and square shapes, the cell movement follow a purse-string mechanism/the mixed pattern. Thus, the present study suggested that for faster wound healing, the cell migration along the wound edge may be found beneficial, and the external healing agent in the form of engineered protein hydrogel accelerate the healing accordingly.

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.

Turning a Luffa Protein into a Self-Assembled Biodegradable Nanoplatform for Multitargeted Cancer Therapy.

The peptide-derived self-assembly platform has attracted increasing attention for its great potential to develop into multitargeting nanomedicines as well as its inherent biocompatibility and biodegradability. However, their clinical application potentials are often compromised by low stability, weak membrane penetrating ability, and limited functions. Herein, inspired by a natural protein from the seeds of Luffa cylindrica, we engineered via epitope grafting and structure design a hybrid peptide-based nanoplatform, termed Lupbin, which is capable of self-assembling into a stable superstructure and concurrently targeting multiple protein-protein interactions (PPIs) located in cytoplasm and nuclei. We showed that Lupbin can efficiently penetrate cell membrane, escape from early endosome-dependent degradation, and subsequently disassemble into free monomers with wide distribution in cytosol and nucleus. Importantly, Lupbin abrogated tumor growth and metastasis through concurrent blockade of the Wnt/ß-catenin signaling and reactivation of the p53 signaling, with a highly favorable in vivo biosafety profile. Our strategy expands the application of self-assembled nanomedicines into targeting intercellular PPIs, provides a potential nanoplatform with high stability for multitargeted cancer therapy, and likely reinvigorates the development of peptide-based therapeutics for the treatment of different human diseases including cancer.

Tunable assembly of protein-microdomains in living vertebrate embryos.

Subcellular events such as trafficking and signaling are regulated by self-assembled protein complexes inside the cell. The ability to rapidly and reversibly manipulate these protein complexes would likely enhance studies of their mechanisms and their roles in biological function and disease manifestation.[1, 2] This manuscript reports that thermally-responsive elastin-like polypeptides (ELPs) linked to fluorescent proteins can regulate the self-assembly and disassembly of protein microdomains within the individual cells of zebrafish embryos. By exploring a library of fluorescent ELP proteins, this reports demonstrates that ELPs can co-assemble different fluorescent proteins inside of embryos. By tuning ELP length and sequence, fluorescent protein microdomains can be assembled at different temperatures, in varying sizes, or for desired periods of time. For the first time in a multicellular living embryo, these studies demonstrate that temperature-mediated ELP assembly can reversibly manipulate assembly of subcellular protein complexes, which may have applications in the study and manipulation of in vivo biological functions.

Probing the Role of Integrins in Keratinocyte Migration Using Bioengineered Extracellular Matrix Mimics.

Bioengineered extracellular matrix (ECM) mimetic materials have tunable properties and can be engineered to elicit desirable cellular responses for wound repair and tissue regeneration. By incorporating relevant cell-instructive domains, bioengineered ECM mimics can be designed to provide well-defined ECM-specific cues to influence cell motility and differentiation. More importantly, bioengineered ECM surfaces are ideal platforms for studying cell-material interactions without the need to genetically alter the cells. Here, we showed that bioengineered ECM mimics can be employed to clarify the role of integrins in keratinocyte migration. Particularly, the roles of α5ß1 and α3ß1 in keratinocytes were examined, given their known importance in keratinocyte motility. Two recombinant proteins were constructed; each protein contains a functional domain taken from fibronectin (FN-mimic) and laminin-332 (LN-mimic), designed to bind α5ß1 and α3ß1, respectively. We examined how patient-derived primary human keratinocytes migrate when sparsely seeded as well as when allowed to move collectively. We found, consistently, that FN-mimic promoted cell migration while the LN-mimic did not support cell motility. We showed that, when keratinocytes utilize α5ß1 integrins on FN-mimics, they were able to form stable focal adhesion plaques and stabilized lamellipodia. On the other hand, keratinocytes on LN-mimic utilized primarily α3ß1 integrins for migration and, strikingly, cells were unable to activate Rac1 and form stable focal adhesion plaques. Taken together, employment of our bioengineered mimics has allowed us to clarify the roles of α5ß1 and α3ß1 integrins in keratinocyte migration, as well as further provided a mechanistic explanation for their differences.