Regulation of EGF-stimulated activation of the PI-3K/AKT pathway by exocyst-mediated exocytosis.

The phosphoinositide-3 kinase (PI-3K)/AKT cell survival pathway is an important pathway activated by EGFR signaling. Here we show, that in addition to previously described critical components of this pathway, i.e., the docking protein Gab1, the PI-3K/AKT pathway in epithelial cells is regulated by the exocyst complex, which is a vesicle tether that is essential for exocytosis. Using live-cell imaging, we demonstrate that PI(3,4,5)P3 levels fluctuate at the membrane on a minutes time scale and that these fluctuations are associated with local PI(3,4,5)P3 increases at sites where recycling vesicles undergo exocytic fusion. Supporting a role for exocytosis in PI(3,4,5)P3 generation, acute promotion of exocytosis by optogenetically driving exocyst-mediated vesicle tethering up-regulates PI(3,4,5)P3 production and AKT activation. Conversely, acute inhibition of exocytosis using Endosidin2, a small-molecule inhibitor of the exocyst subunit Exo70 (also designated EXOC7), or inhibition of exocyst function by siRNA-mediated knockdown of the exocyst subunit Sec15 (EXOC6), impairs PI(3,4,5)P3 production and AKT activation induced by EGF stimulation of epithelial cells. Moreover, prolonged inhibition of EGF signaling by EGFR tyrosine kinase inhibitors results in spontaneous reactivation of AKT without a concomitant relief of EGFR inhibition. However, this reactivation can be negated by acutely inhibiting the exocyst. These experiments demonstrate that exocyst-mediated exocytosis-by regulating PI(3,4,5)P3 levels at the plasma membrane-subserves activation of the PI-3K/AKT pathway by EGFR in epithelial cells.

Endosome motility defects revealed at super-resolution in live cells using HIDE probes.

We report new lipid-based, high-density, environmentally sensitive (HIDE) probes that accurately and selectively image endo-lysosomes and their dynamics at super-resolution for extended times. Treatment of live cells with the small molecules DiIC16TCO or DiIC16'TCO followed by in situ tetrazine ligation reaction with the silicon-rhodamine dye SiR-Tz generates the HIDE probes DiIC16-SiR and DiIC16'-SiR in the endo-lysosomal membrane. These new probes support the acquisition of super-resolution videos of organelle dynamics in primary cells for more than 7 min with no detectable change in endosome structure or function. Using DiIC16-SiR and DiIC16'-SiR, we describe direct evidence of endosome motility defects in cells from patients with Niemann-Pick Type-C disease. In wild-type fibroblasts, the probes reveal distinct but rare inter-endosome kiss-and-run events that cannot be observed using confocal methods. Our results shed new light on the role of NPC1 in organelle motility and cholesterol trafficking.

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells.

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in bacterial and mammalian cells, tagged with fluorescent proteins or membrane-permeable organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

Diverse p53/DNA binding modes expand the repertoire of p53 response elements.

The tumor suppressor protein p53 acts as a transcription factor, binding sequence-specifically to defined DNA sites, thereby activating the expression of genes leading to diverse cellular outcomes. Canonical p53 response elements (REs) are made of two decameric half-sites separated by a variable number of base pairs (spacers). Fifty percent of all validated p53 REs contain spacers between 1 and 18 bp; however, their functional significance is unclear at present. Here, we show that p53 forms two different tetrameric complexes with consensus or natural REs, both with long spacers: a fully specific complex where two p53 dimers bind to two specific half-sites, and a hemispecific complex where one dimer binds to a specific half-site and the second binds to an adjacent spacer sequence. The two types of complexes have comparable binding affinity and specificity, as judged from binding competition against bulk genomic DNA. Structural analysis of the p53 REs in solution shows that these sites are not bent in both their free and p53-bound states when the two half-sites are either abutting or separated by spacers. Cell-based assay supports the physiological relevance of our findings. We propose that p53 REs with long spacers comprise separate specific half-sites that can lead to several different tetrameric complexes. This finding expands the universe of p53 binding sites and demonstrates that even isolated p53 half-sites can form tetrameric complexes. Moreover, it explains the manner in which p53 binds to clusters of more than one canonical binding site, common in many natural REs.

Long-Term Live-Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI-SiR.

Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 s temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 s temporal resolution. HIDE probes such as DiI-SiR are non-toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super-resolution over biologically relevant timescales.

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding.

The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils.

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA.

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg(2+)) and calcium (Ca(2+)) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3' to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg(2+) ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K(+)) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K(d)) for TATA increases in the order K(+ )< Na(+ )< Ca(2+ )< Mg(2+). Except for Mg(2+), K(d) for TATA relative to ATAT is independent of ion concentration, whereas for Mg(2+) the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg(2+), additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.

Exocyst complex mediates recycling of internal cilia.

Primary cilia are slender, cellular antennae that sense extracellular stimuli, and their absence or dysfunction plays a role in numerous human diseases. Prior work has indicated a role of the exocyst tethering complex in cilia biogenesis and maintenance,1-6 with the underlying paradigm that the exocyst targets vesicles to the ciliary base to deliver ciliary cargoes.7-9 However, the role of the exocyst vis-à-vis to primary cilia in living cells and during stimulation is unknown. Herein, using advanced imaging and quantitative analysis reveals that serum stimulation increases the exocyst's localization to cilia by three-fold. This serum-stimulated localization is highly dynamic, and FRAP experiments show that exocysts at the cilia are highly mobile (60%-80%). Super resolution imaging reveals that the xocyst extends past the cilia base to the entire ciliary pocket. To visualize cilia exocytosis, we conducted live cell imaging with pH-sensitive cilia reporters in combination with extracellular pH switching. Strikingly, we observed that an exocyst-positive internal cilia fuses with the cell surface. These live cell results support a novel and dynamic role of the exocyst complex in the delivery of internalized cilia to the cell surface. Moreover, they suggest a novel pathway may be used to recycle primary cilia to the cell surface that engages the exocyst in response to stimuli. This new remarkable plasticity in cilia presence on the surface in response to extracellular stimuli suggest new means to potentially modulate cilia signaling.

An active tethering mechanism controls the fate of vesicles.

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.

Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags.

Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons-by orders of magnitude-than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.

S-Palmitoylation Sorts Membrane Cargo for Anterograde Transport in the Golgi.

While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted, and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by S-palmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargos concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.

α-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle.

Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD.

Organochlorine pesticides (DDTs and HCHs) in soils from the outskirts of Beijing, China.

Concentrations of HCH (hexachlorocyclohexane) and DDT (Dichlorodiphenyltrichloroethane) were determined in shallow subsurface (5-30 cm depth) and deep soil layers (150-180 cm depth) from the outskirts of Beijing, China. Concentrations of total HCHs (including alpha, beta, gamma, delta-isomers) and total DDTs (including p,p'-DDT, p,p'-DDE, p,p'-DDD, o,p'-DDT) in shallow subsurface soils ranged from 1.36 to 56.61 ng/g dw (median 5.25 ng/g), and from 0.77 to 2178 ng/g (median 38.66 ng/g), respectively, and those in the deeper layers were approximately an order of magnitude less. The spatial distribution of HCHs and DDTs reflected the known historical usage of these pesticides. No correlation between the concentrations of pesticides and soil organic matter content or clay content can be found. The factors affecting residue levels and compositions of DDT and HCH were discussed. The contour maps of beta/gamma ratios and DDT/DDE ratios for both the shallow subsurface and deep layer soils were drawn.

Characterization of aliphatic hydrocarbons in deep subsurface soils near the outskirts of Beijing, China.

Thirty-nine deep subsurface soils (150-180 cm depth) near the outskirts of Beijing were investagated. The concentrations including n-alkanes from C13 to C36, pristane and phytane were in the range of 0.60 to 170.10 microg/g, with a median value of 4.26. Carbon preference index values for n-alkanes ranged from 1.08 to 2.98, with a median value of 1.48. The percentage contribution of "wax" n-alkanes was in the range of 6.03%--46.22%. A predominance of odd/even carbon n-alkanes and unresolved complex mixtures with different shapes and ranges were frequently observed. Factor analysis reduced the data set into three principal components and confirming contributions from low (19.58%), medium (20.49%) molecular weight species and long-chain n-alkanes (43.41%), respectively. Molecular biomarkers such as pristane, phytane, hopanes and steranes were detected. Based on the principal component analysis, the concentration profiles and molecular markers, it was found that the aliphatic hydrocarbons were from both biogenic and anthropogenic sources.

Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis.

Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins couple their stage-wise folding/assembly to rapid exocytosis of neurotransmitters in a Munc18-1-dependent manner. The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD, and LD, respectively). We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1.

Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy.

Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.

DNA translocation of ATP-dependent chromatin remodeling factors revealed by high-resolution optical tweezers.

ATP-dependent chromatin remodeling complexes (remodelers) use the energy of ATP hydrolysis to regulate chromatin structures by repositioning and reconfiguring nucleosomes. Ensemble experiments have suggested that remodeler ATPases are DNA translocases, molecular motors capable of processively moving along DNA. This concept of DNA translocation has become a foundation for understanding the molecular mechanisms of ATP-dependent chromatin remodeling and its biological functions. However, quantitative characterizations of DNA translocation by representative remodelers are rare. Furthermore, it is unclear how a unified theory of chromatin remodeling is built upon this foundation. To address these problems, high-resolution optical tweezers have been applied to investigate remodeler translocation on bare DNA and nucleosomal DNA substrates at a single-molecule level. Our strategy is to hold two ends of a single DNA molecule and measure remodeler translocation by detecting the end-to-end extension and tension changes of the DNA molecule in response to chromatin remodeling. These single-molecule assays can reveal detailed kinetics of remodeler translocation, including velocity, processivity, stall force, pauses, direction changes, and even step size. Here we describe instruments, reagents, sample preparations, and detailed protocols for the single-molecule experiments. We show that optical tweezer force microscopy is a powerful and friendly tool for studies of chromatin structures and remodeling.

Single reconstituted neuronal SNARE complexes zipper in three distinct stages.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here, we used optical tweezers to observe in a cell-free reconstitution experiment in real time a long-sought SNARE assembly intermediate in which only the membrane-distal amino-terminal half of the bundle is assembled. Our findings support the zippering hypothesis, but suggest that zippering proceeds through three sequential binary switches, not continuously, in the amino- and carboxyl-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force that mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 k(B)T (where k(B) is Boltzmann's constant and T is temperature) to mediate fusion.

Transverse tunneling through DNA hydrogen bonded to an electrode.

Guanidinium ions tethered to an electrode form electrical contacts to DNA via hydrogen bonding with the backbone phosphates, thus providing a sequence-independent electrical connector for native DNA submerged in an aqueous electrolyte. DNA adlayers on a guanidinium modified electrode can be imaged by scanning tunneling microscopy with tens of pS gap conductance. The image resolution suggests that multiatom contacts contribute to the tunnel conductance, so we estimate that the single-nucleotide pair conductance may be on the order of 1 pS.

Predicting indirect readout effects in protein-DNA interactions.

Recognition of DNA by proteins relies on direct interactions with specific DNA-functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortions induced by proteins. Predicting indirect readout requires knowledge of the variations in DNA curvature and flexibility in the affected region, which we have determined for a series of DNA-binding sites for the E2 regulatory protein by using the cyclization kinetics method. We examined 16 sites containing different noncontacted spacer sequences, which vary by more than three orders of magnitude in binding affinity. For 15 of these sites, the variation in affinity was predicted within a factor of 3, by using experimental curvature and flexibility values and a statistical mechanical theory. The sole exception was traced to differential magnesium ion binding.