Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher "SpyLock". Using "BiLockCatcher", a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.

SpyDisplay: A versatile phage display selection system using SpyTag/SpyCatcher technology.

Phage display is an established method for the in vitro selection of recombinant antibodies and other proteins or peptides from gene libraries. Here we describe SpyDisplay, a phage display method in which the display is achieved via SpyTag/SpyCatcher protein ligation instead of genetically fusing the displayed protein to a phage coat protein. In our implementation, SpyTagged antibody antigen-binding fragments (Fabs) are displayed via protein ligation on filamentous phages carrying SpyCatcher fused to the pIII coat protein. A library of genes encoding Fab antibodies was cloned in an expression vector containing an f1 replication origin, and SpyCatcher-pIII was separately expressed from a genomic locus in engineered E. coli. We demonstrate the functional, covalent display of Fab on phage, and rapidly isolate specific high-affinity clones via phage panning, confirming the robustness of this selection system. SpyTagged Fabs, the direct outcome of the panning campaign, are compatible with modular antibody assembly using prefabricated SpyCatcher modules and can be directly tested in diverse assays. Furthermore, SpyDisplay streamlines additional applications that have traditionally been challenging for phage display: we show that it can be applied to N-terminal display of the protein of interest and it enables display of cytoplasmically folding proteins exported to periplasm via the TAT pathway.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from in vitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E. coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.

Controlled growth of filamentous fatty acid vesicles under flow.

The earliest forms of cellular life would have required a membrane compartment capable of growth and division. Fatty acid vesicles are an attractive model of protocell membranes, as they can grow into filamentous vesicles that readily divide while retaining their contents. In order to study vesicle growth, we have developed a method for immobilizing multilamellar fatty acid vesicles on modified glass surfaces and inducing filamentous membrane growth under flow. Filament formation strictly depended on the presence of freshly neutralized fatty acid micelles in the flow chamber. Using light microscopy, we observed a strong dependence of initial growth velocity on initial vesicle size, suggesting that new fatty acid molecules were incorporated into the membrane over the entire external surface of the vesicle. We examined the influences of flow rate, fatty acid concentration, and salt concentration on filamentous growth and observed drastic shape changes, including membrane pearling, of preexisting membrane tubules in response to osmotic stress. These results illustrate the versatility of flow studies for exploring the process of fatty acid vesicle growth following exposure to free fatty acids.

Rapid RNA exchange in aqueous two-phase system and coacervate droplets.

Compartmentalization in a prebiotic setting is an important aspect of early cell formation and is crucial for the development of an artificial protocell system that effectively couples genotype and phenotype. Aqueous two-phase systems (ATPSs) and complex coacervates are phase separation phenomena that lead to the selective partitioning of biomolecules and have recently been proposed as membrane-free protocell models. We show in this study through fluorescence recovery after photobleaching (FRAP) microscopy that despite the ability of such systems to effectively concentrate RNA, there is a high rate of RNA exchange between phases in dextran/polyethylene glycol ATPS and ATP/poly-L-lysine coacervate droplets. In contrast to fatty acid vesicles, these systems would not allow effective segregation and consequent evolution of RNA, thus rendering these systems ineffective as model protocells.

Micropattern-controlled local microtubule nucleation, transport, and mesoscale organization.

Microtubule organization in living cells is determined by spatial control of microtubule nucleation, their dynamic properties, and transport by molecular motors. Here, we establish a new micropattern-guided method for controlling local microtubule nucleation by spatially confined immobilization of a microtubule polymerase and show that these nucleated microtubules can be transported and organized in space by motor proteins. This assay provides a new platform for deciphering the principles underlying mesoscale microtubule organization.

Directional switching of the kinesin Cin8 through motor coupling.

Kinesin motor proteins are thought to move exclusively in either one or the other direction along microtubules. Proteins of the kinesin-5 family are tetrameric microtubule cross-linking motors important for cell division and differentiation in various organisms. Kinesin-5 motors are considered to be plus-end-directed. However, here we found that purified kinesin-5 Cin8 from budding yeast could behave as a bidirectional kinesin. On individual microtubules, single Cin8 motors were minus-end-directed motors, whereas they switched to plus-end-directed motility when working in a team of motors sliding antiparallel microtubules apart. This kinesin can thus change directionality of movement depending on whether it acts alone or in an ensemble.

Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14.

During cell division, different molecular motors act synergistically to rearrange microtubules. Minus end-directed motors are thought to have a dual role: focusing microtubule ends to poles and establishing together with plus end-directed motors a balance of force between antiparallel microtubules in the spindle. We study here the competing action of Xenopus laevis kinesin-14 and -5 in vitro in situations in which these motors with opposite directionality cross-link and slide microtubules. We find that full-length kinesin-14 can form microtubule asters without additional factors, whereas kinesin-5 does not, likely reflecting an adaptation to mitotic function. A stable balance of force is not established between two antiparallel microtubules with these motors. Instead, directional instability is generated, promoting efficient motor and microtubule sorting. A nonmotor microtubule cross-linker can suppress directional instability but also impedes microtubule sorting, illustrating a conflict between stability and dynamicity of organization. These results establish the basic organizational properties of these antagonistic mitotic motors and a microtubule bundler.

Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, and +TIP dynamics.

Microtubule cytoskeleton function depends on the dynamic interplay of microtubules and various microtubule-binding proteins. To gain an understanding of cytoskeleton function at the molecular level, it is important to measure quantitatively how cytoskeletal proteins interact with each other in space and time. Here we describe fluorescence microscopy-based in vitro assays on chemically functionalized glass slides for the study of several aspects of microtubule cytoskeleton dynamics: single motor movements, dynamic microtubule plus-end tracking, antiparallel microtubule sliding by microtubule-crosslinking motors, and microtubule gliding by surface-immobilized motors. The combination of a passivating polyethylene glycol layer on the glass with covalently attached functional groups for selective protein capturing ensures excellent control of the surface properties and good preservation of protein activities in these assays. Common to all assays is that they can be performed in the presence of high concentrations of soluble proteins or even cell extract, which in combination with total internal reflection fluorescence microscopy allows the study of complex protein mixtures that were previously not accessible to quantitative imaging in vitro.

Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro and in Xenopus egg extract spindles.

BACKGROUND: Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE: These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.

Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles.

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein-dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.