Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions.

Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

In stressed cells, the ADAR1p110 isoform is phosphorylated and translocated from the nucleus to the cytoplasm, where it protects transcripts with 3′-UTR dsRNA structures from Staufen1-mediated decay, thus suppressing cellular apoptosis. Both p150 and p110 isoforms of ADAR1 convert adenosine to inosine in double-stranded RNA (dsRNA). ADAR1p150 suppresses the dsRNA-sensing mechanism that activates MDA5–MAVS–IFN signaling in the cytoplasm. In contrast, the biological function of the ADAR1p110 isoform, which is usually located in the nucleus, is largely unknown. Here, we show that stress-activated phosphorylation of ADAR1p110 by MKK6–p38–MSK MAP kinases promotes its binding to Exportin-5 and its export from the nucleus. After translocating to the cytoplasm, ADAR1p110 suppresses apoptosis in stressed cells by protecting many antiapoptotic gene transcripts that contain 3′-untranslated-region dsRNA structures primarily comprising inverted Alu repeats. ADAR1p110 competitively inhibits binding of Staufen1 to the 3′-untranslated-region dsRNAs and antagonizes Staufen1-mediated mRNA decay. Our study reveals a new stress-response mechanism in which human ADAR1p110 and Staufen1 regulate surveillance of a set of mRNAs required for survival of stressed cells.