RESUMO
Cell-cell interfaces are found throughout multicellular organisms, from transient interactions between motile immune cells to long-lived cell-cell contacts in epithelia. Studies of immune cell interactions, epithelial cell barriers, neuronal contacts and sites of cell-cell fusion have identified a core set of features shared by cell-cell interfaces that critically control their function. Data from diverse cell types also show that cells actively and passively regulate the localization, strength, duration and cytoskeletal coupling of receptor interactions governing cell-cell signalling and physical connections between cells, indicating that cell-cell interfaces have a unique membrane organization that emerges from local molecular and cellular mechanics. In this Review, we discuss recent findings that support the emerging view of cell-cell interfaces as specialized compartments that biophysically constrain the arrangement and activity of their protein, lipid and glycan components. We also review how these biophysical features of cell-cell interfaces allow cells to respond with high selectivity and sensitivity to multiple inputs, serving as the basis for wide-ranging cellular functions. Finally, we consider how the unique properties of cell-cell interfaces present opportunities for therapeutic intervention.
Assuntos
Comunicação Celular/fisiologia , Compartimento Celular/fisiologia , Fenômenos Fisiológicos Celulares/fisiologia , Animais , Fusão Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Mecanotransdução Celular/fisiologia , Neurônios/citologia , Neurônios/fisiologiaRESUMO
Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis.
Assuntos
Clatrina , Endocitose , Animais , Clatrina/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mamíferos/metabolismoRESUMO
During developmental processes and wound healing, activation of living cells occurs with spatiotemporal precision and leads to rapid release of soluble molecular signals, allowing communication and coordination between neighbors. Nonliving systems capable of similar responsive release hold great promise for information transfer in materials and site-specific drug delivery. One nonliving system that offers a tunable platform for programming release is synthetic cells. Encased in a lipid bilayer structure, synthetic cells can be outfitted with molecular conduits that span the bilayer and lead to material exchange. While previous work expressing membrane pore proteins in synthetic cells demonstrated content exchange, user-defined control over release has remained elusive. In mammalian cells, connexon nanopore structures drive content release and have garnered significant interest since they can direct material exchange through intercellular contacts. Here, we focus on connexon nanopores and present activated release of material from synthetic cells in a light-sensitive fashion. To do this, we re-engineer connexon nanopores to assemble after post-translational processing by a protease. By encapsulating proteases in light-sensitive liposomes, we show that assembly of nanopores can be triggered by illumination, resulting in rapid release of molecules encapsulated within synthetic cells. Controlling connexon nanopore activity provides an opportunity for initiating communication with extracellular signals and for transferring molecular agents to the cytoplasm of living cells in a rapid, light-guided manner.
Assuntos
Células Artificiais , Nanoporos , Canais Iônicos , Lipossomos , PorinasRESUMO
The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.
Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Polissacarídeos/química , Anticorpos , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Epitopos , Imunofluorescência , Células HEK293 , Humanos , Lectinas , Bicamadas Lipídicas , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Polissacarídeos/metabolismo , Domínios ProteicosRESUMO
Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here, we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted claudin-4 alone can form adhesive membrane interfaces without the accessory proteins that are present in vivo By controlling the molecular composition of the inner and outer leaflets of jetted vesicle membranes, we show that claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins with ectodomains as small as 5â nm but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of claudins and their small size can contribute to the polarization of epithelial cells.
Assuntos
Membrana Celular/metabolismo , Claudina-4/metabolismo , Proteolipídeos/metabolismo , Junções Íntimas/metabolismo , Claudina-4/genética , Células Epiteliais/metabolismo , Humanos , Lipossomas Unilamelares/metabolismoRESUMO
Branching morphogenesis in the mammary gland is achieved by the migration of epithelial cells through a microenvironment consisting of stromal cells and extracellular matrix (ECM). Here we show that galectin-1 (Gal-1), an endogenous lectin that recognizes glycans bearing N-acetyllactosamine (LacNAc) epitopes, induces branching migration of mammary epithelia in vivo, ex vivo, and in 3D organotypic cultures. Surprisingly, Gal-1's effects on mammary patterning were independent of its glycan-binding ability and instead required localization within the nuclei of mammary epithelia. Nuclear translocation of Gal-1, in turn, was regulated by discrete cell-surface glycans restricted to the front of the mammary end buds. Specifically, α2,6-sialylation of terminal LacNAc residues in the end buds masked Gal-1 ligands, thereby liberating the protein for nuclear translocation. Within mammary epithelia, Gal-1 localized within nuclear Gemini bodies and drove epithelial invasiveness. Conversely, unsialylated LacNAc glycans, enriched in the epithelial ducts, sequestered Gal-1 in the extracellular environment, ultimately attenuating invasive potential. We also found that malignant breast cells possess higher levels of nuclear Gal-1 and α2,6-SA and lower levels of LacNAc than nonmalignant cells in culture and in vivo and that nuclear localization of Gal-1 promotes a transformed phenotype. Our findings suggest that differential glycosylation at the level of tissue microanatomy regulates the nuclear function of Gal-1 in the context of mammary gland morphogenesis and in cancer progression.
Assuntos
Núcleo Celular/metabolismo , Galectina 1/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Neoplasias Mamárias Animais/etiologia , Morfogênese , Polissacarídeos/fisiologia , Animais , Feminino , Glicosilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
The generation of homogeneously glycosylated proteins is essential for defining glycoform-specific activity and improving protein-based therapeutics. We present a novel glycodendron prosthetic which can be site-selectively appended to recombinant proteins to create 'N-glycosylated' glycoprotein mimics. Using computational modeling, we designed the dendrimer scaffold and protein attachment point to resemble the native N-glycan architecture. Three piperidine-melamine glycodendrimers were synthesized via a chemoenzymatic route and attached to human growth hormone and the Fc region of human IgG. These products represent a new class of engineered biosimilars bearing novel glycodendrimer structures.
Assuntos
Dendrímeros/química , Piperidinas/química , Polissacarídeos/química , Proteínas/química , Dendrímeros/síntese química , Humanos , Estrutura Molecular , Triazinas/químicaRESUMO
Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP's influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality and multiplexing. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into structures to control cell and tissue shape and behavior.
Assuntos
Citoesqueleto de Actina , Actinas , Proteínas dos Microfilamentos , Ligação Proteica , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Citoesqueleto de Actina/metabolismo , Humanos , Animais , Cinética , Engenharia de Proteínas/métodosRESUMO
A general platform for the safe and effective oral delivery of biologics would revolutionize the administration of protein-based drugs, improving access for patients and lowering the financial burden on the health-care industry. Because of their dimensions and physiochemical properties, nanomaterials stand as promising vehicles for navigating the complex and challenging environment in the gastrointestinal (GI) tract. Recent developments have led to materials that protect protein drugs from degradation and enable controlled release in the small intestine, the site of absorption for most proteins. Yet, once present in the small intestine, the protein must transit through the secreted mucus and epithelial cells of the intestinal mucosa into systemic circulation, a process that remains a bottleneck for nanomaterial-based delivery. One attractive pathway through the intestinal mucosa is the paracellular route, which avoids cell trafficking and other degradative processes in the interior of cells. Direct flux between cells is regulated by epithelial tight junctions (TJs) that seal the paracellular space and prevent protein flux. Here, we describe a smart nanoparticle system that directly and transiently disrupts TJs for improved protein delivery, an unrealized goal to-date. We take inspiration from enteropathogenic bacteria that adhere to intestinal epithelia and secrete inhibitors that block TJ interactions in the local environment. To mimic these natural mechanisms, we engineer nanoparticles (EnteroPatho NPs) that attach to the epithelial glycocalyx and release TJ modulators in response to the intestinal pH. We show that EnteroPatho NPs lead to TJ disruption and paracellular protein delivery, giving rise to a general platform for oral delivery.
Assuntos
Nanopartículas , Junções Íntimas , Humanos , Nanopartículas/química , Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Proteínas/química , Proteínas/metabolismo , Mucosa Intestinal/metabolismo , Portadores de Fármacos/química , Células CACO-2 , AnimaisRESUMO
As biologics used in the clinic outpace the number of new small molecule drugs, an important challenge for their efficacy and widespread use has emerged, namely tissue penetrance. Macromolecular drugs - bulky, high-molecular weight, hydrophilic agents - exhibit low permeability across biological barriers. Epithelial and endothelial layers, for example within the gastrointestinal tract or at the blood-brain barrier, present the most significant obstacle to drug transport. Within epithelium, two subcellular structures are responsible for limiting absorption: cell membranes and intercellular tight junctions. Previously considered impenetrable to macromolecular drugs, tight junctions control paracellular flux and dictate drug transport between cells. Recent work, however, has shown tight junctions to be dynamic, anisotropic structures that can be targeted for delivery. This review aims to summarize new approaches for targeting tight junctions, both directly and indirectly, and to highlight how manipulation of tight junction interactions may help usher in a new era of precision drug delivery.
Assuntos
Junções Intercelulares , Junções Íntimas , Humanos , Junções Íntimas/metabolismo , Junções Intercelulares/metabolismo , Epitélio/metabolismo , Transporte Biológico , Sistemas de Liberação de Medicamentos , Preparações Farmacêuticas/metabolismoRESUMO
In metazoans, living cells achieve capabilities beyond individual cell functionality by assembling into multicellular tissue structures. These higher-order structures represent dynamic, heterogeneous, and responsive systems that have evolved to regenerate and coordinate their actions over large distances. Recent advances in constructing micrometer-sized vesicles, or synthetic cells, now point to a future where construction of synthetic tissue can be pursued, a boon to pressing material needs in biomedical implants, drug delivery systems, adhesives, filters, and storage devices, among others. To fully realize the potential of synthetic tissue, inspiration has been and will continue to be drawn from new molecular findings on its natural counterpart. In this review, we describe advances in introducing tissue-scale features into synthetic cell assemblies. Beyond mere complexation, synthetic cells have been fashioned with a variety of natural and engineered molecular components that serve as initial steps toward morphological control and patterning, intercellular communication, replication, and responsiveness in synthetic tissue. Particular attention has been paid to the dynamics, spatial constraints, and mechanical strengths of interactions that drive the synthesis of this next-generation material, describing how multiple synthetic cells can act as one.
Assuntos
Células Artificiais , Engenharia Tecidual , Comunicação Celular , Sistemas de Liberação de MedicamentosRESUMO
Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP's influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into new structures to control cell and tissue shape and behavior.
RESUMO
The galectin family of glycan-binding proteins is thought to mediate many cellular processes by oligomerizing cell surface glycoproteins and glycolipids into higher-order aggregates. This hypothesis reflects the known oligomeric states of the galectins themselves and their binding properties with multivalent ligands in vitro, but direct evidence of their ability to cross-link ligands on a cell surface is lacking. A major challenge in fundamental studies of galectin-ligand interactions is that their natural ligands comprise a heterogeneous collection of glycoconjugates that share related glycan structures but disparate underlying scaffolds. Consequently, there is no obvious means to selectively monitor the behaviors of natural galectin ligands on live cell surfaces. Here we describe an approach for probing the galectin-induced multimerization of glycoconjugates on cultured cells. Using RAFT polymerization, we synthesized well-defined glycopolymers (GPs) functionalized with galectin-binding glycans along the backbone, a lipid group on one end and a fluorophore on the other. After insertion into live cell membranes, the GPs' fluorescence lifetime and diffusion time were measured in the presence and absence of galectin-1. We observed direct evidence for galectin-1-mediated extended cross-linking on the engineered cells, a phenomenon that was dependent on glycan structure. This platform offers a new approach to exploring the "galectin lattice" hypothesis and to defining galectin ligand specificity in a physiologically relevant context.
Assuntos
Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Galectinas/metabolismo , Glicoconjugados/metabolismo , Animais , Células CHO , Membrana Celular/química , Cricetinae , Galectina 1/análise , Galectina 1/metabolismo , Galectinas/análise , Glicoconjugados/análise , Polimerização , Ligação ProteicaRESUMO
Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the electroactive bacterium Shewanella oneidensis for metabolic and genetic control over Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring S. oneidensis cells. The reaction progress and kinetics were manipulated by tailoring the central carbon metabolism. Similarly, EET-CuAAC activity was dependent on specific EET pathways that could be regulated via inducible expression of EET-relevant proteins: MtrC, MtrA, and CymA. EET-driven CuAAC exhibited modularity and robustness in the ligand and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without regeneration, something inaccessible to traditional chemical reductants. Finally, S. oneidensis enabled bioorthogonal CuAAC membrane labeling on live mammalian cells without affecting cell viability, suggesting that S. oneidensis can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.
RESUMO
The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.
Assuntos
Citoesqueleto de Actina/metabolismo , Multimerização Proteica , Utrofina/metabolismo , Citoesqueleto de Actina/química , Fatores de Despolimerização de Actina/metabolismo , Actinas , Animais , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia Intravital , Cinética , Microscopia de Fluorescência , Mutação , Neuropeptídeos/metabolismo , Mutação Puntual , Ligação Proteica/genética , Domínios Proteicos/genética , Coelhos , Imagem Individual de Molécula , Utrofina/química , Utrofina/genéticaRESUMO
In vertebrates, epithelial permeability is regulated by the tight junction (TJ) formed by specialized adhesive membrane proteins, adaptor proteins, and the actin cytoskeleton. Despite the TJ's critical physiological role, a molecular-level understanding of how TJ assembly sets the permeability of epithelial tissue is lacking. Here, we identify a 28-amino-acid sequence in the TJ adaptor protein ZO-1, which is responsible for actin binding, and show that this interaction is essential for TJ permeability. In contrast to the strong interactions at the adherens junction, we find that the affinity between ZO-1 and actin is surprisingly weak, and we propose a model based on kinetic trapping to explain how affinity could affect TJ assembly. Finally, by tuning the affinity of ZO-1 to actin, we demonstrate that epithelial monolayers can be engineered with a spectrum of permeabilities, which points to a promising target for treating transport disorders and improving drug delivery.
Assuntos
Junções Aderentes/metabolismo , Polaridade Celular/fisiologia , Epitélio/metabolismo , Junções Íntimas/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , PermeabilidadeRESUMO
Surface topography is a key parameter in regulating the morphology and behavior of single cells. At multicellular level, coordinated cell displacements drive many biological events such as embryonic morphogenesis. However, the effect of surface topography on collective migration of epithelium has not been studied in detail. Mastering the connection between surface features and collective cellular behaviour is highly important for novel approaches in tissue engineering and repair. Herein, we used photopatterned microtopographies on azobenzene-containing materials and showed that smooth topographical cues with proper period and orientation can efficiently orchestrate cell alignment in growing epithelium. Furthermore, the experimental system allowed us to investigate how the orientation of the topographical features can alter the speed of wound closure in vitro. Our findings indicate that the extracellular microenvironment topography coordinates their focal adhesion distribution and alignment. These topographic cues are able to guide the collective migration of multicellular systems, even when cell-cell junctions are disrupted.
Assuntos
Técnicas de Cultura de Células/instrumentação , Células Epiteliais/citologia , Animais , Compostos Azo/química , Movimento Celular , Colágeno/química , Cães , Adesões Focais , Técnicas de Inativação de Genes , Junções Intercelulares , Células Madin Darby de Rim Canino , Propriedades de Superfície , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1-CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective "openness" of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution "openness," suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties.