A microfluidic platform for the synthesis of polymer and polymer-protein-based protocells.

In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.

The Clinical Observership Program: A Valuable Experience for Oral & Maxillofacial Surgery Residents.

PURPOSE: Training during oral and maxillofacial surgery residency must include exposure to the scope of the specialty, but success in practice often requires particular experience and knowledge of complex oral regenerative procedures such as bone grafting and implant surgery, as well as practice management. Osteo Science Foundation created the Clinical Observership Program (COP) in 2017 to provide residents the opportunity to spend several weeks in an established oral and maxillofacial surgery practice to increase experience in these areas. The purpose of this study is to report the results of a survey of all resident participants in the COP from 2017 to 2021 in which participants were asked to rate their experience numerically. MATERIALS AND METHODS: This is an institutional retrospective case series completed via an electronic survey sent to all participants in the COP from 2017 to 2021. The primary outcome is the subjective assessment of the COP based on six questions in which the respondent was asked to rate the program on a scale of 1 to 10 (10 being best). Categories included: 1) Did the program achieve expectations? 2) Was adequate time spent with the mentor? 3) Did you observe/participate in a variety of procedures? 4) Did the mentor provide additional didactic education? 5) Did you learn about practice management? and 6) How would you rate the overall experience? Descriptive statistics including mean score and standard deviation of each question were calculated, and no other covariates were analyzed. RESULTS: All 55 participants in the COP from 2017 to 2021 were contacted and 55 complete responses were received. The overall mean score for all categories rated by the residents was 9.63, the mean rating for questions 1 to 6 were 9.55, 9.89, 9.21, 9.60, 9.69, and 9.86 respectively, and the range of scores was 7 to 10. CONCLUSION: Overall, residents rated the COP experience highly. This survey indicates that the COP is a valuable supplemental experience in oral and maxillofacial surgery resident education.

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior.

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

Dental prosthesis extraoral indexing and pick-up technique for mandibular full arch immediate rehabilitation in fibula free flap reconstruction.

Same-day ablative and reconstructive surgeries for the treatment of head and neck pathologies are gaining in popularity with the recognition that single-day surgeries reduce morbidity and increase quality of life. Implant-borne prosthetics on the donor graft provide immediate dental reconstruction. This report describes a novel technique for extraoral pickup of a full arch immediate prosthesis from the donor site free flap. This technique minimizes intraoperative occlusal adjustments, saves intraoperative time, prevents undesirable "rolling" of a fibula segment, and immediately rehabilitates patients with dental prosthetics.

Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences.

Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid-liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Here, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over phase separation of RGG domains fused to FRB and FKBP in the presence of dimerizer. Further, using a photocaged dimerizer, we achieve optically induced condensation both in cell-sized emulsions and within live cells. Collectively, these modular tools allow multiple strategies to promote phase separation of a common core IDR for tunable control of condensate assembly.

LFA-1 signals to promote actin polymerization and upstream migration in T cells.

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the phosphoinositide 3-kinase (PI3K) and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, whereas VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl (also known as CBL), failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior.This article has an associated First Person interview with the first author of the paper.

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates.

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or "membraneless organelles" such as nucleoli, stress granules, and P-bodies. Because of their propensity to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of a novel enzymatic activity in protein coacervates with the light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of Caenorhabditis elegans LAF-1, we functionalized condensates with enzymatic activity in vitro and show that enzyme localization to coacervates enhances assembly and activity of split enzymes. To build condensates that function as light-emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and displayed significantly increased reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. Because our model condensates have low viscosities, we predict NanoLuc diffusion-limited behavior in most other condensates and thus propose the condensate-Nanoluc system as a potential strategy for high-throughput screening of condensate targeting drugs. By splitting the NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. In addition, we demonstrate control of the spatial localization of the enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity and that this activity can be spatially and temporally controlled via biochemical reconstitution and design of protein surfactants.

Immediate Teeth in Fibulas: Expanded Clinical Applications and Surgical Technique.

PURPOSE: The placement of immediate implants and teeth during jaw reconstruction using a fibula free flap has increased in recent years. Modifications of traditional fibula reconstructive techniques are needed to maximize success. This technique has not been described in patients requiring simultaneous soft tissue reconstruction. Our patient cohort includes cases with malignant pathology and those requiring skin paddles.  With digital workflows and point-of-care 3D printing, surgery is no longer delayed weeks for prosthesis fabrication. The purpose of this case series is to demonstrate a single institution's experience with expanded clinical applications and surgical techniques that enable predictable outcomes for immediate teeth in fibula flaps. MATERIALS AND METHODS: Ninety-five implants were placed in 22 patients undergoing fibula reconstruction of the jaw with immediate implants and an immediate dental prosthesis. Skin paddles were used in 10 patients while 12 patients had native mucosa. Six patients were treated for malignancies and underwent postoperative radiation. Implant success and complications were compared between implants with skin paddles and implants with native mucosa. RESULTS: Of 95 implants, 92 implants integrated for a 97% integration rate. All 13 radiated implants in 4 patients integrated. All 36 implants adjacent to skin paddles in 10 patients integrated. Seven implants were lost in a delayed fashion 9 to 15 months postoperatively resulting in a 93% overall implant success rate. Of the 22 patients, diagnoses were benign pathology for 11 patients, malignant pathology for 6 patients, gunshot wounds for 3 patients, and osteoradionecrosis for 2 patients. CONCLUSION: Immediate placement of dental prostheses on immediate implants during fibula reconstruction of the jaws can be performed with a high rate of predictability. This technique can be expanded to select patients needing skin paddles. Modifications of traditional fibula reconstructive techniques are helpful to minimize soft tissue and prosthetic challenges.

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes.

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3'-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity.

Migration against the direction of flow is LFA-1-dependent in human hematopoietic stem and progenitor cells.

The recruitment of immune cells during inflammation is regulated by a multi-step cascade of cell rolling, activation, adhesion and transmigration through the endothelial barrier. Similarly, hematopoietic stem and progenitor cells (HSPCs) use this pathway to migrate and home to the bone marrow. After selectin-mediated braking, HSPCs migrate on adhesion ligands presented by the vascular endothelium including ICAM-1, VCAM-1 or MAdCAM-1. Here, we report that both the KG1a stem cell line and primary bone marrow CD34+ HSPCs can migrate against the direction of fluid flow on surfaces coated with cell adhesion molecules (CAMs), a behavior thus far only reported in T lymphocytes. We demonstrate that KG1a cells and primary HSPCs migrate upstream on surfaces presenting ICAM-1, downstream on surfaces presenting VCAM-1, and both upstream and downstream on surfaces presenting MAdCAM-1. In addition, we demonstrate that KG1a cells and HSPCs display upstream migration both on surfaces with multiple CAMs, as well as on human umbilical vein endothelial cell (HUVEC) monolayers. By blocking with monoclonal antibodies, we show that lymphocyte function-associated antigen-1 (LFA-1) is the key receptor responsible for upstream migration on the endothelium during the trafficking of HSPCs to the bone marrow.This article has an associated First Person interview with the first author of the paper.

A simulation of the random and directed motion of dendritic cells in chemokine fields.

Dendritic cells (DCs) are the most effective professional antigen-presenting cell. They ferry antigen from the extremities to T cells and are essential for the initiation of an adaptive immune response. Despite interest in how DCs respond to chemical stimuli, there have been few attempts to model DC migration. In this paper, we simulate the motility of DCs by modeling the generation of forces by filopodia and a force balance on the cell. The direction of fliopodial extension is coupled to differential occupancy of cognate chemokine receptors across the cell. Our model simulates chemokinesis and chemotaxis in a variety of chemical and mechanical environments. Simulated DCs undergoing chemokinesis were measured to have a speed of 5.1 ± 0.07 µm·min-1 and a persistence time of 3.2 ± 0.46 min, consistent with experiment. Cells undergoing chemotaxis exhibited a stronger chemotactic response when exposed to lower average chemokine concentrations, also consistent with experiment. We predicted that when placed in two opposing gradients, cells will cluster in a line, which we call the "line of equistimulation;" this clustering has also been observed. We calculated the effect of varying gradient steepness on the line of equistimulation, with steeper gradients resulting in tighter clustering. Moreover, gradients are found to be most potent when cells are in a gradient of chemokine whose mean concentration is close to the binding of the Kd to the receptor, and least potent when the mean concentration is 0.1Kd. Comparing our simulations to experiment, we can give a quantitative measure of the strength of certain chemokines relative to others. Assigning the signal of CCL19 binding CCR7 a baseline strength of 1, we found CCL21 binding CCR7 had a strength of 0.28, and CXCL12 binding CXCR4 had a strength of 0.30. These differences emerge despite both chemokines having virtually the same Kd, suggesting a mechanism of signal amplification in DCs requiring further study.

The cancer glycocalyx mechanically primes integrin-mediated growth and survival.

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

Immediate Teeth in Fibulas: Planning and Digital Workflow With Point-of-Care 3D Printing.

PURPOSE: Point-of-care 3-dimensional (3D) printing has become more common in recent years because many hospitals have created 3D printing laboratories. Traditional techniques to fabricate an immediate dental prosthesis for fibula and implant reconstructions have involved outsourcing to dental laboratories. This results in delays, making it suitable only for benign disease. In the present report, we have demonstrated a technique for in-house creation of a 3D printed dental prosthesis for placement of implants at free fibula maxillofacial reconstruction. Our digital method has reduced costs and shortened the interval to surgery compared with traditional laboratory techniques. MATERIALS AND METHODS: Twelve patients underwent free fibula reconstruction of the mandible or maxilla with immediate implants and immediate teeth. A dental implant-retained restoration was created before surgery for immediate placement at fibula reconstruction. For the first 5 patients, the prosthesis was fabricated by a dental laboratory after virtual surgical planning. For the next 7 patients, the prosthesis was designed by the surgeon and 3D printed via the in-house laboratory. Four of these in-house cases were performed for malignant disease with skin paddles. RESULTS: All 12 patients received an immediate implant-retained fixed prosthesis at fibula reconstruction. The time required to generate the in-house 3D printed prostheses was significantly shorter than that required to create the dental laboratory-fabricated prostheses. The costs were also less with the 3D printed prostheses compared with the dental laboratory-fabricated prostheses. CONCLUSIONS: The digital workflow we have presented eliminates the delay in creating a dental laboratory-fabricated provisional dental prosthesis for fibula and implant reconstruction. This allows for immediate dental restoration for patients with malignant disease previously considered unsuitable owing to the inherent delay required using an offsite dental laboratory. A decrease in cost to create in-house 3D printed prostheses was noted compared with the prostheses fabricated by a dental laboratory. Case selection is critical to predict the soft tissue needs for composite defects.

Janus dendrimersomes coassembled from fluorinated, hydrogenated, and hybrid Janus dendrimers as models for cell fusion and fission.

A three-component system of Janus dendrimers (JDs) including hydrogenated, fluorinated, and hybrid hydrogenated-fluorinated JDs are reported to coassemble by film hydration at specific ratios into an unprecedented class of supramolecular Janus particles (JPs) denoted Janus dendrimersomes (JDSs). They consist of a dumbbell-shaped structure composed of an onion-like hydrogenated vesicle and an onion-like fluorinated vesicle tethered together. The synthesis of dye-tagged analogs of each JD component enabled characterization of JDS architectures with confocal fluorescence microscopy. Additionally, a simple injection method was used to prepare submicron JDSs, which were imaged with cryogenic transmission electron microscopy (cryo-TEM). As reported previously, different ratios of the same three-component system yielded a variety of structures including homogenous onion-like vesicles, core-shell structures, and completely self-sorted hydrogenated and fluorinated vesicles. Taken together with the JDSs reported herein, a self-sorting pathway is revealed as a function of the relative concentration of the hybrid JD, which may serve to stabilize the interface between hydrogenated and fluorinated bilayers. The fission-like pathway suggests the possibility of fusion and fission processes in biological systems that do not require the assistance of proteins but instead may result from alterations in the ratios of membrane composition.

Human Neutrophils Will Crawl Upstream on ICAM-1 If Mac-1 Is Blocked.

The recruitment of neutrophils to sites of inflammatory insult is a hallmark of the innate immune response. Neutrophil recruitment is regulated by a multistep process that includes cell rolling, activation, adhesion, and transmigration through the endothelium commonly referred to as the leukocyte adhesion cascade. After selectin-mediated braking, neutrophils migrate along the activated vascular endothelium on which ligands, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are expressed. Previous studies have shown that two cells that commonly home from blood vessel to tissue-T cells and hematopoietic stem and progenitor cells-use the integrin lymphocyte functional antigen-1 (LFA-1) to migrate against the direction of shear flow once adherent on ICAM-1 surfaces. Like T cells and hematopoietic stem and progenitor cells, neutrophils express LFA-1, but they also express macrophage-1 antigen (Mac-1), which binds to ICAM-1. Previous reports have shown that neutrophils will not migrate against the direction of flow on ICAM-1, but we hypothesized this was due to the influence of Mac-1. Here, we report that both the HL-60 neutrophil-like cell line and primary human neutrophils can migrate against the direction of fluid flow on ICAM-1 surfaces via LFA-1 if Mac-1 is blocked; otherwise, they migrate downstream. We demonstrate this both on ICAM-1 surfaces and on activated endothelium. In sum, both LFA-1 and Mac-1 binding ICAM-1 play a critical role in determining the direction of neutrophil migration along the endothelium, and their interaction may play an important role in controlling neutrophil trafficking during inflammation.

Enhanced Cell Killing by Paclitaxel-Loaded Recombinant Protein Micelles Bearing Integrin-Binding and Cell-Penetrating Peptides.

Peptide ligands are effective and specific vectors that can target cell surface receptors, and have shown great potential for targeting drug delivery vehicles. Often, materials used as drug delivery matrices are chemically synthesized and difficult to functionalize, which compromises their development as smart drug carriers. Here, we assemble carriers from a recombinant protein as a novel approach to overcome these limitations. We have previously shown that oleosin, a natural surfactant protein, can be engineered to self-assemble into spherical micelles, and that functionalizing oleosin with RGDS can increase cellular uptake in integrin bearing cells. Here, we investigated whether we could further enhance cellular by incorporating either a RGDS synergy peptide PHSRN or a cell-penetrating Tat peptide derived from human immunodeficiency virus (HIV). The resulting modified oleosins self-assemble into spherical micelles in aqueous environments. Spherical micelles made from oleosin can effectively encapsulate the hydrophobic chemotherapeutic drug paclitaxel (PX). After 15 hours, 350 nM PX loaded oleosin micelles equipped with both RGDS and Tat increased cell killing by twofold compared to free paclitaxel, and 1.2-fold compared to micelles made from RGD-oleosin alone. Micelles equipped with PHSRN alone does not facilitate cell killing compared to free paclitaxel, whereas micelles equipped with both PHSRN and RGDS increased cell killing by 1.1 fold compared to micelles with RGDS alone in 15 hours. Therefore, incorporating multiple motifs into oleosin is an approach for candidate for making a versatile drug delivery carrier.

Engineering the Echogenic Properties of Microfluidic Microbubbles Using Mixtures of Recombinant Protein and Amphiphilic Copolymers.

Microbubbles are used as ultrasound contrast agents in medical diagnosis and also have shown great promise in ultrasound-mediated therapy. However, short lifetime and broad size distribution of microbubbles limit their applications in therapy and imaging. Moreover, it is challenging to tailor the echogenic response of microbubbles to make them suitable for specific applications. To overcome these challenges, we use microfluidic flow-focusing to prepare monodisperse microbubbles with a mixture of a recombinant amphiphilic protein, oleosin, and a synthetic amphiphilic copolymer, Pluronic. We show that these microbubbles have superior uniformity and stability under ultrasonic stimulation compared to commercial agents. We also demonstrate that by using different Pluronics, the echogenic response of the microbubbles can be tailored. Our work shows the versatility of using the combination of microfluidics and protein/copolymer mixtures as a method of engineering microbubbles. This tunability could potentially be important and powerful in producing microbubble agents for theranostic applications.

Bioactive cell-like hybrids coassembled from (glyco)dendrimersomes with bacterial membranes.

A library of amphiphilic Janus dendrimers including two that are fluorescent and one glycodendrimer presenting lactose were used to construct giant dendrimersomes and glycodendrimersomes. Coassembly with the components of bacterial membrane vesicles by a dehydration-rehydration process generated giant cell-like hybrid vesicles, whereas the injection of their ethanol solution into PBS produced monodisperse nanometer size assemblies. These hybrid vesicles contain transmembrane proteins including a small membrane protein, MgrB, tagged with a red fluorescent protein, lipopolysaccharides, and glycoproteins from the bacterium Escherichia coli. Incorporation of two colored fluorescent probes in each of the components allowed fluorescence microscopy to visualize and demonstrate coassembly and the incorporation of functional membrane channels. Importantly, the hybrid vesicles bind a human galectin, consistent with the display of sugar moieties from lipopolysaccharides or possibly glycosylated membrane proteins. The present coassembly method is likely to create cell-like hybrids from any biological membrane including human cells and thus may enable practical application in nanomedicine.

Onion-like glycodendrimersomes from sequence-defined Janus glycodendrimers and influence of architecture on reactivity to a lectin.

A library of eight amphiphilic Janus glycodendrimers (GDs) with d-mannose (Man) headgroups, a known routing signal for lectin-mediated transport processes, was constructed via an iterative modular methodology. Sequence-defined variations of the Janus GD modulate the surface density and sequence of Man after self-assembly into multilamellar glycodendrimersomes (GDSs). The spatial mode of Man presentation is decisive for formation of either unilamellar or onion-like GDS vesicles. Man presentation and Janus GD concentration determine GDS size and number of bilayers. Beyond vesicle architecture, Man topological display affects kinetics and plateau level of GDS aggregation by a tetravalent model lectin: the leguminous agglutinin Con A, which is structurally related to endogenous cargo transporters. The agglutination process was rapid, efficient, and readily reversible for onion-like GDSs, demonstrating their value as versatile tools to explore the nature of physiologically relevant glycan/lectin pairing.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.