Marc C. Llaguno

Phase transitions in the assembly of multivalent signalling proteins

Cells are organized on length scales ranging from ångström to micrometres. However, the mechanisms by which ångström-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid–liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott–Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.

Massive calcium-activated endocytosis without involvement of classical endocytic proteins.

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4–64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP2) synthesis: PIP2 substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5′-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP2 induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP2 when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP2 and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A2 inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and loss of reversible FM 4–64 binding confirm that 40–75% of the cell surface is internalized. Ongoing MEND stops in 2–4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and β-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces.

Placing and shaping liposomes with reconfigurable DNA nanocages

The diverse structure and regulated deformation of lipid bilayer membranes are among a cells most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

Chemically functionalized carbon films for single molecule imaging

Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and chemical linkage to introduce a high density of bioactive ligands onto nanometer-thick carbon films and enable selective enrichment of individual macromolecular complexes at subnanogram levels. The introduced ligands are physically separated. Ni-NTA, Protein G and DNA/RNA oligonucleotides were covalently linked to the carbon surface. They embody negligible mass and their stability makes the functionalized films able to survive long-term storage and tolerate variations in pH, temperature, salts, detergents, and solvents. We demonstrated the application of the new method to the electron microscopic imaging of the substrate-bound C3PO, an RNA-processing enzyme important for the RNA interference pathway. On the ssRNA-linked carbon surface, the formation of C3PO oligomers at subnanomolar concentrations likely mimics their assembly onto ssRNA substrates presented by their native partners. Interestingly, the 3D reconstructions by negative stain EM reveal a side port in the C3PO/ssRNA complex, and the 15Å cryoEM map showed extra density right above the side port, which probably represents the ssRNA. These results suggest a new way for ssRNAs to interact with the active sites of the complex. Together our data demonstrate that the surface-engineered carbon films are suitable for selectively enriching low-abundance biological complexes at nanomolar level and for developing novel applications on a large number of surface-presented molecules.