Simple Host-Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way

Three-Dimensional Nanocrystal Superlattices Grown in Nanoliter Microfluidic Plugs

We studied the self-assembly of inorganic nanocrystals (NCs) confined inside nanoliter droplets (plugs) into long-range ordered superlattices. We showed that a capillary microfluidic platform can be used for the optimization of growth conditions for NC superlattices and can provide insights into the kinetics of the NC assembly process. The utility of our approach was demonstrated by growing large (up to 200 μm) three-dimensional (3D) superlattices of various NCs, including Au, PbS, CdSe, and CoFe(2)O(4). We also showed that it is possible to grow 3D binary nanoparticle superlattices in the microfluidic plugs

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

m6A RNA methylation regulates the UV-induced DNA damage response

Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response (DDR), detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 minutes) and transiently induced at DNA damage sites in response to UV. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL31 and the demethylase FTO2. In the absence of METTL3 catalytic activity, cells showed delayed repair of UV-induced cyclobutane pyrimidine (CPD) adducts and elevated sensitivity to UV, demonstrating the importance of m6A in the UV-responsive DDR. Multiple DNA polymerases are involved in the UV response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair (NER) pathway3, while others participate in trans-lesion synthesis (TLS) to allow replication past damaged lesions in S phase4. DNA polymerase κ (Pol κ), which has been implicated in both NER and TLS5,6, required the catalytic activity of METTL3 for immediate localization to UV-induced DNA damage sites. Importantly, Pol κ over-expression qualitatively suppressed the CPD removal defect associated with METTL3 loss. Taken together, we have uncovered a novel function for RNA m6A modification in the UV-induced DDR, and our findings collectively support a model whereby m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival

Identification of Smo N-linked glycosylation sites.

<p><b>A</b>. A multiple sequence alignment of Smo proteins from different phyla are shown. Consensus sequences for N-linked glycosylation are highlighted in gray and the Asn acceptor residues are bold. Sites conserved across vertebrate proteins are indicated as N1-N4. The predicted <i>D</i>. <i>melanogaster</i> sites are not tightly conserved with vertebrates. <b>B</b>. Drosophila Smo is N-glycosylated. Cell lysates prepared from Cl8 cells expressing Hh with wild type or NQ5 dSmo proteins were treated with the indicated deglycosylating enzymes. Wild type Smo demonstrated ER (arrow) and post-ER (arrowhead) glycosylation species. NQ5 migrated similarly to the fully deglycosylated species under all conditions (arrow). <b>C</b>. Mouse Smo is N-glycosylated. Cellular lysates from <i>Smo-/-</i> cells stably expressing mSmoWT or mSmoNQ4 were treated with deglycosylating enzymes and subjected to SDS-PAGE and western blot. mSmoWT resolves as two distinct forms (lane 2). The arrow marks the ER form and the arrowhead indicates the post-ER form. mSmoNQ4 migrates in SDS-PAGE similarly to the PNGase-treated wild type protein (lanes 4–5). <b>C’</b>. mSmoNQ4 is O-glycosylated. Lysates were prepared from NIH3T3 cells expressing mSmoNQ4 and subjected to lambda phosphatase, PNGase and O-glycosidase/neuraminidase treatments. The upper band collapsed upon O-glycosidase/neuraminidase treatment. <b>D</b>. Expression of individual N to Q dSmo mutants. The indicated N to Q dSmo mutants were expressed in Cl8 cells and cell lysates were analyzed by SDS-PAGE and western blot against the Myc tag. Kin serves as loading control. <b>E</b>. Extracellular mSmo consensus sites are N-glycosylated. Mutation of individual extracellular mSmo glycosylation sites induced faster mobility on SDS-PAGE. mSmoN450Q migrated similarly to SmoWT. For western blots, mSmo was detected using anti-Smo and dSmo with anti-Myc. Kinesin (Kin) and Tubulin (Tub) were blotted for loading controls.</p

N-glycosylation status correlates with signal bias.

<p>A model for modulation of mSmo signal bias. SAG (yellow) binding to N-glycosylated mSmo stabilizes a conformation that effectively signals through both canonical and non-canonical routes. SAG binding to the mSmo mutant stripped of N-glycans (red) silences non-canonical signaling, but is highly permissive for canonical signaling.</p

N213 glycosylation partially compensates for N336 N-glycan loss.

<p><b>A.</b> The indicated mutants were expressed in Cl8 cells. Each of the mutant proteins migrated more quickly in SDS-PAGE than wild type Smo, but not as quickly as SmoNQ4 or NQ5. <b>B.</b> Glycosylation at N213 partially compensates for N336 glycan loss. The rescue reporter assay was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2A</a>. Each of the indicated double mutants, with the exception of N213Q,N336Q, was able to rescue reporter gene expression in the <i>smo</i> knockdown background to a level similar to that of N336Q. dSmoN213Q,N336Q demonstrated a level of activity similar to dSmoNQ5. Significance was determined using Student’s t-test. <b>C.</b> N336Q-containing double mutants are retained in the ER. Cl8 cells expressing the indicated Smo proteins (anti-Myc, magenta), the Cal-EGFP-KDEL marker (green) and Hh were examined by immunofluorescence microscopy. Whereas wild type Smo reached the plasma membrane, the double mutants overlapped with the ER marker. ActinRed (red) marks F-actin. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). <b>D.</b> Treatment of lysates from WT or N213Q,N336Q expressing cells with deglycosylating enzymes reveals that the N213,336Q mutant is present in the EndoH sensitive ER fraction, arrowhead. <b>E-E’</b>. N336Q and N213Q,N336Q mutants have disulfide bond defects. Biotin-maleimide was used to tag free thiol groups in cellular lysates prepared from Cl8 cells expressing WT, N336Q, N213Q,N336Q and C320A dSmo proteins. WT dSmo is not well captured on NeutrAvadin beads (lane 2, bound). N-glycan mutants are captured similarly to the disulfide bond mutant C320A (lanes 3–5, bound), indicating that at least one disulfide bridge is disrupted by N-glycan loss. E’ shows the ratio of bound to unbound dSmo proteins normalized to kinesin. Relative binding was determined by densitometry analysis of two independent binding assays. C320A, which has an established disulfide bond defect served as positive control. It’s binding ratio was arbitrarily set to 1.0 and other values are shown relative to it. Error bars are provided to show the standard deviation between the two experiments. <b>F.</b> dSmoN213Q,N336Q fails to rescue <i>smo</i> knockdown <i>in vivo</i>. <i>UAS-dsmoN213Q</i>, <i>N336Q</i> was co-expressed with <i>UAS-dicer</i> and <i>UAS-smo</i>^{<i>3’UTR</i>} using the <i>nubbin-Gal4</i> driver. Its expression did not modify the <i>smo</i> knockdown phenotype (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2F</a>). Multiple progeny were analyzed over two crosses and a representative wing is shown.</p

N-linked glycans are required for dSmo trafficking and activity.

<p><b>A.</b> dSmoNQ5 does not signal <i>in vitro</i>. Cl8 cells were transfected with control or <i>smo 5’UTR</i> dsRNA, the <i>ptcΔ136-luciferase</i> reporter, <i>pAc-renilla</i> control, <i>pAc-myc-smoWT</i> or <i>NQ5</i>, and <i>pAc-hh</i> or empty vector control. Hh-induced reporter activity (gray bars) was ablated by knockdown of endogenous <i>smo</i> and rescued by dSmo cDNA lacking UTR sequence for wild type, but not for dSmoNQ5. <b>B-B’</b>. dSmoNQ5 demonstrates altered sub-cellular localization. Cl8 cells expressing Calreticulin-EGFP-KDEL ER marker (GFP-ER, green) and Myc-SmoWT or NQ5 in the presence or absence of Hh were imaged by immunofluorescence microscopy. Wild type dSmo (anti-Myc, magenta) localized to puncta that did not overlap with the ER marker in the absence of Hh, and translocated to the plasma membrane in response to Hh. The NQ5 mutant overlapped with the ER marker under both conditions. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right box). <b>B’</b>. GFP-ER colocalizes with V5 tagged BiP, Calnexin (Cnx) and Calreticulin (Crc). DAPI marks the nucleus. <b>C-H.</b> dSmoNQ5 does not signal <i>in vivo</i>. Transgenes encoding wild type (G) or NQ5 (H) dSmo proteins were expressed in the <i>nubbin>dicer;smo</i>^{<i>3’UTR</i>} background (E). Whereas wild type Smo could rescue the loss of function phenotype induced by <i>smo</i>^{<i>3’UTR</i>}, dSmoNQ5 could not (G-H compared to F and C-D, control). <i>UAS-EGFP</i> was expressed in the <i>nubbin>dicer;smo</i>^{<i>3’UTR</i>} background and serves as a control for normalized transgene dosage (F). <b>I.</b> Wild type and NQ5 dSmo proteins are present at similar protein levels in wing imaginal disc tissue lysate. The dSmoN213Q,N336Q protein level is higher.</p