Boxin Xue

Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.

Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.

Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space

Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein–protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB–EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB–EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB–EF-Tu interactions.

Vesicle Size Regulates Nanotube Formation in the Cell

Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling.

Multicolor Super-resolution Fluorescence Microscopy with Blue and Carmine Small Photoblinking Polymer Dots

Advances in the development of small photoblinking semiconducting polymer dots (Pdots) have attracted great interest for use in super-resolution microscopy. However, multicolor super-resolution imaging using conventional small photoblinking Pdots remains a challenge due to their limited color choice, broad emission spectrum, and heavy spectrum crosstalk. Here, we introduce two types of small photoblinking Pdots with different colors and relatively narrow emission spectra: blue PFO Pdots and carmine PFTBT5 Pdots for blinking-based statistical nanoscopy. Both of these probes feature ultrahigh single-particle brightness, very strong photostability, superior biocompatibility, and robust fluorescence fluctuation. In addition, these small photoblinking Pdots serve as excellent labels for dual-color super-resolution optical fluctuation imaging (SOFI) of specific subcellular structures, indicating their promise for long-term multicolor SOFI nanoscopy with high spatiotemporal resolution.

Live Cell Visualization of Multiple Protein–Protein Interactions with BiFC Rainbow

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

Live-cell and super-resolution imaging reveal that the distribution of wall-associated protein A is correlated with the cell chain integrity of Streptococcus mutans

Streptococcus mutans is a primary pathogen responsible for dental caries. It has an outstanding ability to form biofilm, which is vital for virulence. Previous studies have shown that knockout of Wall-associated protein A (WapA) affects cell chain and biofilm formation of S. mutans. As a surface protein, the distribution of WapA remains unknown, but it is important to understand the mechanism underlying the function of WapA. This study applied the fluorescence protein mCherry as a reporter gene to characterize the dynamic distribution of WapA in S. mutans via time-lapse and super-resolution fluorescence imaging. The results revealed interesting subcellular distribution patterns of WapA in single, dividing and long chains of S. mutans cells. It appears at the middle of the cell and moves to the poles as the cell grows and divides. In a cell chain, after each round of cell division, such dynamic relocation results in WapA distribution at the previous cell division sites, resulting in a pattern where WapA is located at the boundary of two adjacent cell pairs. This WapA distribution pattern corresponds to the breaking segmentation of wapA deletion cell chains. The dynamic relocation of WapA through the cell cycle increases our understanding of the mechanism of WapA in maintaining cell chain integrity and biofilm formation.

Intranucleus Single-Molecule Imaging in Living Cells

Many critical processes occurring in mammalian cells are stochastic and can be directly observed at the single-molecule level within their physiological environment, which would otherwise be obscured in an ensemble measurement. There are various fundamental processes in the nucleus, such as transcription, replication, and DNA repair, the study of which can greatly benefit from intranuclear single-molecule imaging. However, the number of such studies is relatively small mainly because of lack of proper labeling and imaging methods. In the past decade, tremendous efforts have been devoted to developing tools for intranuclear imaging. Here, we mainly describe the recent methodological developments of single-molecule imaging and their emerging applications in the live nucleus. We also discuss the remaining issues and provide a perspective on future developments and applications of this field.

Superior performance with sCMOS over EMCCD in super-resolution optical fluctuation imaging.

Abstract. Super-resolution optical fluctuation imaging (SOFI) is a fast and low-cost live-cell optical nanoscopy for extracting subdiffraction information from the statistics of fluorescence intensity fluctuation. As SOFI is based on the fluctuation statistics, rather than the detection of single molecules, it poses unique requirements for imaging detectors, which still lack a systematic evaluation. Here, we analyze the influences of pixel sizes, frame rates, noise levels, and different gains in SOFI with simulations and experimental tests. Our analysis shows that the smaller pixel size and faster readout speed of scientific-grade complementary metal oxide semiconductor (sCMOS) enables SOFI to achieve high spatiotemporal resolution with a large field-of-view, which is especially beneficial for live-cell super-resolution imaging. Overall, as the performance of SOFI is relatively insensitive to the signal-to-noise ratio (SNR), the gain in pixel size and readout speed exceeds the loss in SNR, indicating sCMOS is superior to electron multiplying charge coupled device in context to SOFI in many cases. Super-resolution imaging of cellular microtubule structures with high-order SOFI is experimentally demonstrated at large field-of-view, taking advantage of the large pixel number and fast frame rate of sCMOS cameras.