Xiaolin Nan

Organelle tracking in a living cell with microsecond time resolution and nanometer spatial precision

The study of cellular processes such as organelle transport often demands particle tracking with microsecond time-resolution and nanometer spatial precision, posing significant challenges to existing tracking methods. Here, we have developed a novel strategy for two-dimensional tracking of gold nanoparticles (GNPs) with 25 mus time resolution and approximately 1.5 nm spatial precision, by using a quadrant photodiode to record the positions of GNPs in an objective-type dark-field microscope. In combination with a feedback loop, this technique records long, high time-resolution and spatial precision trajectories of endocytosed GNPs transported by the molecular motors kinesin and dynein in a living cell. In the full range of organelle velocities (0-8 microm s(-1)), we clearly resolve the individual 8 nm steps of cargoes carried by kinesin, and the 8, 12, 16, 20, and 24 nm steps of those carried by dynein. These experiments yield new information about molecular motor stepping in living cells.

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

Significance Rat sarcoma (Ras) proteins play central roles in both normal and oncogenic signaling. Mechanisms of how Ras interacts with its effectors on the cell membrane, however, are still poorly understood, significantly hampering efforts to target this molecule in human cancer. Here we have used quantitative superresolution fluorescence microscopy in combination with carefully engineered biological systems to show that Ras dimers drive oncogenic signaling through the Raf-MAPK pathway. Our study suggests a new, dimer model of Ras-Raf signaling and the potential value of Ras dimers as a therapeutic target. Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling

Significance This paper describes a quantitative imaging approach based on photoactivated localization microscopy for precise protein localization and stoichiometric analysis inside an intact cell. With this approach, we directly resolved individual protein monomers, dimers, and multimers in fixed mammalian cells. We used this technique to study the dimerization and multimerization of the protein kinase RAF, a putative mechanism for mutant Rat sarcoma (RAS)-mediated RAF activation as well as for the paradoxical activation of RAF/MAPK in RAF WT cells and tumors upon treatment with RAF kinase inhibitors. We presented direct evidence that RAF indeed forms dimers and occasionally higher-order multimers under various activating conditions. These observations provide critical insight into the biological regulation of RAF signaling and oncogenesis. The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF–CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.

Chemically assembled single-wall carbon nanotubes and their electrochemistry

Single-wall carbon nanotubes (SWNTs) have attracted great interest because of their unique structural, mechanical, and electronic properties. Recent studies have shown that SWNTs may have potential applications in diverse fields, for example in nanodevices, sensors, and scanning probes. The ability to prepare highly oriented SWNT arrays is crucial for improving the performance of these devices. Recently, Papadimitrakopoulos et al. and our group reported the formation of short SWNT assemblies oriented normal to the substrate through surface reactions. This finding makes the study on the properties of the aligned SWNTs possible. Knowledge of the electrochemical properties of the aligned SWNT assemblies is essential with respect to their potential applications in developing scanning electrochemical microscopy, designing electrochemical and bioelectrochemical sensors, and fabricating ultramicroelectrodes arrays. However, no such studies have been reported up to now. Herein, we report the fabrication and characterization of chemically assembled SWNTs (ca-SWNTs), which are constructed by the combination of a self-assembling procedure and a surface condensation reaction. We demonstrate that ca-SWNTs, though immobilized on insulating monolayer-coated gold substrates, are electrochemically addressable and behave like a collection of closely spaced microelectrodes. Furthermore, metals such as copper can be electrodeposited onto ca-SWNTs. The ca-SWNT preparation strategy, which is different from our previous procedures, is shown in Scheme 1. SWNTs were cut by oxidation in mixed acid under sonication. During the cutting process, the open ends of the SWNTs were modified with COOH groups. The SWNT assemblies were then prepared through a surface condensation reaction between COOH groups at the ends of shortened SWNTs and NH2 groups at preformed 11amino-n-undecylmercaptan (AUM) monolayers on Au substrates with dicyclohexylcarbodiimide (DCC) as condensing agent (DCC-assisted condensation method; Scheme 1). spectroscopy turns out to be an extremely structurally sensitive method. In conclusion, we can state that electron energy-loss spectroscopy has proved to be an excellent experimental device for determining and picturing the structural and electronic details of a compound. Here, in combination with theoretical calculations at the ∫full potential∫ level, it is more convincing than any other method, such as X-ray or neutron diffraction. In the case of two metal diboride ±dicarbides, contradictory notions about the arrangement of boron and carbon atoms could thus be arbitrated by combining the methods of diffraction, DFT calculations, and EELS. Contrary to what was once assumed, CaB2C2 and LaB2C2 are not isotypic.

Intracellular imaging of HCV RNA and cellular lipids by using simultaneous two-photon fluorescence and coherent anti-Stokes Raman scattering microscopies.

The propagation of HCV requires host–virus interactions that support infection, replication, and viral particle assembly. 2] Genotypes 1a and 1b of HCV induce changes in lipid metabolism and cause the formation of endoplasmic reticulum (ER)-derived membranous webs on which HCV replicates. HCV also induces the accumulation of lipid droplets (LDs), known as steatosis, on which certain HCV proteins are known to reside. Currently, there is no method that permits the observation of spatiotemporal relationships between HCV RNA and alterations in host-cell lipids. Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful, multiphoton, vibrational imaging modality that is ideal for imaging lipids in live, unstained cells and tissues. Selective imaging of lipids is easily achieved by tuning the frequency difference between two pulsed excitation lasers to match the vibrational frequency of the C H bonds that are abundant in lipids. The use of pulsed, near-IR excitation sources enables the easy combination of CARS with other nonlinear imaging techniques, such as two-photon fluorescence (TPF) microscopy. Herein we establish methods that combine CARS and TPF microscopies to simultaneously examine the subcellular localization of HCV replicon RNA (Figure 1), a noninfectious cell model for HCV replication, and changes in lipid phenotype in live Huh-7 hepatoma cells. The approach is also applicable to cell culture models for HCV infection. First we investigated the localization of LDs in CARS mi ACHTUNGTRENNUNGcrosACHTUNGTRENNUNGcopy images of Huh-7 cells that were treated with only the DMRIE-C transfection reagent (mock-transfected). These cells contained LDs that dominated the CH2 vibrational resonance and corresponded to a size range of 0.3–2 mm (see Figure S1 in the Supporting Information). However, when Huh-7 cells were transfected with lipoplexes comprising transfection reagent containing HCV RNA from the pFK-I389luc/NS3-3’/5.1 subACHTUNGTRENNUNGgenomic replicon (Figure 1B), 14] we observed a trend for increased lipid density in the living cells exposed to HCV RNA as compared to mock-transfected cells (0.35 0.12 vs. 0.25 0.10 a.u. , respectively) that was consistent with the initiation of changes in lipid metabolism by the HCV replicon RNA (Figure S1). To image HCV RNA by TPF, the replicon RNAs were labeled with a two-photon fluorophore (fluorescein) at either the 5’ end of the positive strands, according to Figure 1C, or along the length of the RNA (see the Supporting Information). For simultaneous imaging with combined CARS and TPF microscopies, we used a 711 nm (2 ps) laser beam as both the pump beam for CARS and the excitation beam for TPF. The fluorescein molecules attached to fully labeled and 5’-labeled HCV RNA in lipoplexes were easily probed by TPF, and the fluorescence was stable over a continuous scan of more than five minutes (Figure S2), perhaps due to solid stacking among lipid and RNA molecules in the lipoplexes. We utilized 5’-labeled RNA to study the localization of HCV RNA in Huh-7 cells because activity studies measuring the luciferase genetic reporter demonstrated that 5’-labeled RNA was replication competent (45 17% activity compared to unlabeled RNA), whereas fully labeled RNA was not. We observed that the HCV RNA–liposome lipoplexes were condensed into tightly packed structures that gave strong TPF signals up to 8 h post-transfection (Figure 2C and D). As previously demonstrated, we observed that the HCV RNA localized to the perinuclear region and on or near to LDs/lipoplexes (Figure 2D). The cells showed a progressive increase in LDs during the first 16 h after transfection, as shown in Figure 2B and D and as quantified in Figure 2E (see the Supporting Information). There was a positive correlation between the density of LDs and the levels of HCV RNA, that is, the cells with the highest density of LDs were also transfected with the highest amount of RNA (Figure 2E). To our knowledge, this is the first detailed, live-cell quantification of total LDs over time in cells expressing HCV RNA and proteins. At 16 h post-transfection, the fluorescence signals were significantly reduced and more diffuse, and, by 24 h, the 5’-labeled RNA was no longer visible by TPF (Figure 2C). These observations are consistent with the half-life of the labeled RNA. Since replication of the labeled RNA did not involve the incorporation of new fluorophores, our ability to image viral RNA was limited to the lifetime of the fluorescently labeled RNA that was initially delivered to the cells. According to luciferase assays of cellular lysates from Huh-7 cells transfected with unlabeled RNA, the luciferase signal was 100-fold higher in cells transfected with viral RNA than in mock-transfected cells at 2–6 h post-transfection (data not shown). This implies that transfected HCV RNA enters the cells and begins dissociating from liposomes within 2–6 h, followed by translation of encoded viral proteins and replication of the viral RNA. Thus, we believe that the significant increase in LDs observed after 16 h can be attributed directly to the effects of fluorescently labeled RNA [a] X. Nan, Prof. X. S. Xie Department of Chemistry and Chemical Biology, Harvard University 12 Oxford Street, Cambridge, MA 02138 (USA) [b] Dr. A. M. Tonary, Prof. A. Stolow, Prof. J. P. Pezacki The Steacie Institute for Molecular Sciences National Research Council of Canada 100 Sussex Drive, Ottawa, K1A 0R6 (Canada) Fax: (+1)613-952-0068 E-mail : John.Pezacki@nrc-cnrc.gc.ca [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Accelerating 3B single-molecule super-resolution microscopy with cloud computing

To the Editor: The recently invented technique of Bayesian localization microscopy1 relaxes the requirement for an extremely high signal-to-background ratio for single-molecule super-resolution microscopy2–5. It enables a standard wide-field fluorescence microscope to generate super-resolution images from the stochastic bleaching and blinking6,7 of fluorescence dyes (3B analysis). Although the new 3B microscopy has enormous potential to put the power of super-resolution imaging into the hands of average biologists, its underlying algorithm can be an impediment. Calculating an area of 50 × 50 pixels takes approximately a full day on a state-of-the-art desktop computer (Fig. 1a). For larger image areas, the need for massive computing resources makes the 3B analysis inaccessible to many labs and limits its scope of application. We report here a simple solution for everyone: the Amazon Elastic Compute Cloud (Amazon EC2). Figure 1 3B analysis using Amazon EC2 Aside from its flexible and cost-effective super-computing power, the greatest advantages of Amazon EC2 are its accessibility and ease of use. Normally, porting a computational code to a new system involves the configuration of a system-dependent software environment. The convoluted configuration process—not to mention the increased complexity for cluster computing—often discourages scientists without programming backgrounds. Use of Amazon EC2 significantly lowers this entry barrier. By packaging the executable program together with its running environment in a single image file known as the Amazon machine image (AMI), we can distribute 3B analysis to the public through the Amazon Marketplace. Hundreds of processes can run on instances, with each instance configured as an exact copy of the AMI. A user can launch massively parallel 3B processes on the Cloud from a local computer in just a few minutes (Fig. 1b and Supplementary Video 1). Large computing power allows data analysis without compromising on field of view. We tested Amazon EC2 using the photoactivatable (PA) mCherry–tagged podosome data from ref. 1 (Fig. 1c). The recorded image was divided into small mosaics of 12 × 12 pixels. The mosaics were analyzed in parallel and recombined to form a super-resolution image of 9.6 × 9.6 µm. Given the same computation time, an eight-thread 3.40-GHz Intel Core i7 desktop computer could analyze a region of 2.715 × 2.715 µm. The image generated from 100 virtual cores from Amazon EC2 was 12 times as large. An entry-level user of the Amazon Cloud may obtain up to 100 instances at about

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM) for Nanoscale Imaging of Protein-Protein Interactions in Cells

0.02–

p21-Activated Kinase (PAK) Regulates Cytoskeletal Reorganization and Directional Migration in Human Neutrophils

0.06 per instance hour. Depending on the market price, reconstruction of a 25 × 25–µm image costs less than

Ras Dimer Formation as a New Signaling Mechanism and Potential Cancer Therapeutic Target.

25. We strongly recommend the use of our bash scripts to continuously monitor the reconstructed image (Supplementary Software and tutorial therein). The calculation should be terminated only when the structrual features have converged, as otherwise potentially serious image artifacts may still be present. Researchers may apply for Amazon Web Service research grants for additional computing hours. We performed a test with 300 instances of 600 virtual cores. An image of PAmCherry-tagged tubulins in U2OS cells (from our own experiments) with the size of 150 pixels × 100 pixels × 1,500 frames could be reconstructed in only 210 min (Fig. 1d), whereas on a state-of-the-art desktop computer, this process would have taken 9 d. Cloud computing has been transforming the information technology industry through its elastic and flexible on-demand services. Although cloud computing has contributed to the success of many companies such as Dropbox and Netflix, until now it has been little used in biological research. Through this Correspondence, we hope to highlight its huge potential for the imaging community.

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.