Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.

The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-positive bacteria. Here, we describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chemically modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, we show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. We find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addition to the protein immobilization and modification tool kit.

Using recombinant amphiphilic proteins to self-assemble suprastructures would allow precise control over surfactant chemistry and the facile incorporation of biological functionality. We used cryo-TEM to confirm self-assembled structures from recombinantly produced mutants of the naturally occurring sunflower protein, oleosin. We studied the phase behavior of protein self-assembly as a function of solution ionic strength and protein hydrophilic fraction, observing nanometric fibers, sheets, and vesicles. Vesicle membrane thickness correlated with increasing hydrophilic fraction for a fixed hydrophobic domain length. The existence of a bilayer membrane was corroborated in giant vesicles through the localized encapsulation of hydrophobic Nile red and hydrophilic calcein. Circular dichroism revealed that changes in nanostructural morphology in this family of mutants was unrelated to changes in secondary structure. Ultimately, we envision the use of recombinant techniques to introduce novel functionality into these materials for biological applications.protein surfactants | self-assembled suprastructure | cryogenic transmission microscopy

CD47 on red blood cells (RBCs) reportedly signals "self" by binding SIRP␣ on phagocytes, at least in mice. Such interactions across and within species, from mouse to human, are not yet clear and neither is the relation to cell adhesion. Using human SIRP␣1 as a probe, antibody-inhibitable binding to CD47 was found only with human and pig RBCs (not mouse, rat, or cow). In addition, CD47-mediated adhesion of human and pig RBCs to SIRP␣1 surfaces resists sustained forces in centrifugation (as confirmed by atomic force microscopy) but only at SIRP␣-coating densities far above those measurable on human neutrophils, monocytes, and THP-1 macrophages. While interactions strengthen with deglycosylation of SIRP␣1, low copy numbers explain the absence of RBC adhesion to phagocytes under physiologic conditions and imply that the interaction being studied is not responsible for red cell clearance in humans. Evidence of clustering nonetheless suggests mechanisms of avidity enhancement. Finally, using the same CD47 antibodies and soluble SIRP␣1, bone marrow-derived mesenchymal stem cells were assayed and found to display CD47 but not bind SIRP␣1 significantly. The results thus demonstrate that SIRP␣-CD47 interactions, which reportedly define self, exhibit cell type specificity and limited cross-species reactivity.

Giant and stable worm micelles formed from poly(ethylene glycol) (PEG)-based diblock copolymer amphiphiles have the potential advantage compared to smaller assemblies for delivery of a large quantity of hydrophobic drugs or dyes per carrier. Here we show that worm micelles can be targeted to cells with internalization and delivery of nontoxic dyes as well as cytotoxic drugs. Constituent copolymers are end-biotinylated to mediate high affinity binding of worm micelles to both avidin-bearing surfaces and biotin-specific receptors on smooth muscle cells. Pristine worm micelles, that lack biotin, show much less frequent and nonspecific point attachments to the same surfaces. Biotinylated worm micelles prove stable in aqueous solution for at least a month and also prove capable of loading, retaining, and delivering hydrophobic dyes and drugs. The results thus demonstrate the feasibility of targeted delivery by polymeric worm micelles.

Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality — demonstrating the robust and facile nature of this reaction.

The reduction in expression of the integral membrane protein CD47 in human red blood cells (RBCs) deficient in protein 4.2 suggests that protein 4.2 may mediate a linkage of CD47 to the membrane skeleton. We compared the fractions of membrane skeleton-attached CD47, Rh-associated glycoprotein (RhAG), Rh, and band 3 in normal and protein 4.2-deficient cells using fluorescence-imaged microdeformation. We found that CD47 attachment decreases from 55% in normal cells to IntroductionThe role of the ubiquitously expressed transmembrane protein CD47 integrin-associated protein (IAP) on various cell types has been the focus of an increasing number of studies. On hematopoietic cells, surface expression of CD47 is involved in leukocyte activation and chemotaxis, 1 T-cell adhesion, 2 and macrophage multinucleation. 3 On mature erythrocytes CD47 interacts with the signal regulatory protein ␣ (SIRP-␣) of splenic macrophages. 4 Subsequent studies with red blood cells (RBCs) from CD47 knockout mice suggest that this association inhibits phagocytosis, thereby delaying erythrocyte clearance from the circulation and implicating CD47 as a "marker of self" in mice. 5 In an attempt to further elucidate the function and interactions of CD47 in the human erythrocyte, we recently provided visual evidence for colocalization of CD47 with Rh and Rh-associated glycoprotein (RhAG) on the RBC surface. 6 The results appear consistent with reduced expression of CD47 in Rh null erythrocytes, which lack Rh and RhAG proteins. 7 It has been shown recently that RBCs expressing no RhCE (DϪϪ erythrocytes) or considerably reduced levels of RhCE (D.. and R N erythrocytes) also have reduced levels of CD47, suggesting that CD47 may be associated preferentially with the RhCE protein. 8 However, our recent studies of Rh null cells concluded that approximately 60% of CD47 on the RBC surface is connected to the membrane skeleton, also referred to in this work as the spectrin-actin cytoskeleton, independent of Rh or RhAG. 6 In addition to cis interactions between CD47 and Rh, protein 4.2 has recently been suggested to have vertical interactions with CD47, linking it to the cytoskeleton in RBCs. Protein 4.2 binds to the cytoplasmic domain of band 3 and to spectrin in solution, suggesting that it stabilizes the connection of band 3 to spectrin mediated by ankyrin. 9 Several different protein 4.2 mutations result in a loss of protein 4.2 expression, causing spherocytosis in the homozygous state, 10 and 2 groups have shown that there is also a substantial reduction of CD47 on protein 4.2-deficient RBCs (4.2-Hammersmith 11 ; 4.2-Lisboa and 4.2-Nancy 8 ). Bruce et al 11 noted that the absence of protein 4.2 in 4.2-deficient Hammersmith RBCs was associated with either a 75% reduction in CD47 using flow cytometry (FC) or an apparent absence of CD47 by Western blot. Mouro-Chanteloup et al 8 also observed an approximately 80% reduction in CD47 in 4.2-deficient Lisboa and Nancy cells by FC. Unlike CD47, expression levels of band 3, RhAG, and Rh peptides in 4.2-deficien...

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