The ciliary lumen accommodates passive diffusion and vesicle-assisted trafficking in cytoplasm-ciliary transport.

Transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Using virtual three-dimensional superresolution light microscopy, the movements of membrane and soluble proteins from the cytoplasm to the primary cilium were mapped. In addition to the well-characterized intraflagellar transport (IFT) route, we found two new pathways within the lumen of the primary cilium: passive diffusion and vesicle-assisted transport routes that are adopted by proteins for cytoplasm-cilium transport in live cells. Through these pathways, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route, and more than half of membrane-embedded G protein-coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside primary cilia. Ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis, and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins.

CLASP2 recognizes tubulins exposed at the microtubule plus-end in a nucleotide state-sensitive manner.

CLASPs (cytoplasmic linker-associated proteins) are ubiquitous stabilizers of microtubule dynamics, but their molecular targets at the microtubule plus-end are not understood. Using DNA origami-based reconstructions, we show that clusters of human CLASP2 form a load-bearing bond with terminal non-GTP tubulins at the stabilized microtubule tip. This activity relies on the unconventional TOG2 domain of CLASP2, which releases its high-affinity bond with non-GTP dimers upon their conversion into polymerization-competent GTP-tubulins. The ability of CLASP2 to recognize nucleotide-specific tubulin conformation and stabilize the catastrophe-promoting non-GTP tubulins intertwines with the previously underappreciated exchange between GDP and GTP at terminal tubulins. We propose that TOG2-dependent stabilization of sporadically occurring non-GTP tubulins represents a distinct molecular mechanism to suppress catastrophe at the freely assembling microtubule ends and to promote persistent tubulin assembly at the load-bearing tethered ends, such as at the kinetochores in dividing cells.

Nucleoplasmic signals promote directed transmembrane protein import simultaneously via multiple channels of nuclear pores.

Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. Most models propose transport through the nuclear pore complexes, but a central outstanding question is whether transit occurs through their central or peripheral channels. Using live-cell high-speed super-resolution single-molecule microscopy we could distinguish protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains containing nuclear localization signals into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels; however, the mutated proteins can still complete their translocation using only the peripheral channels, albeit at a reduced rate. Such proteins can still translocate using only the peripheral channels when central channel is blocked, but blocking the peripheral channels blocks translocation through both channels. This suggests that peripheral channel transport is the default mechanism that was adapted in evolution to include aspects of receptor-mediated central channel transport for directed trafficking of certain membrane proteins.

3D Tracking-Free Approach for Obtaining 3D Super-Resolution Information in Rotationally Symmetric Biostructures.

Currently, it is highly desirable but still challenging to obtain high-resolution (<50 nm) three-dimensional (3D) super-resolution information on structures in fixed specimens as well as for dynamic processes in live cells. Here we introduce a simple approach, without using 3D super-resolution microscopy or real-time 3D particle tracking, to estimate 3D sub-diffraction-limited structural or dynamic information in rotationally symmetric biostructures. This is a postlocalization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability distributions on the basis of prior known structural knowledge. This analysis is ideal in cases where the ultrastructure of a cellular structure is known but the substructural localization of a particular (usually mobile) protein is not. The method has been successfully applied to achieve 3D structural and functional sub-diffraction-limited information for 25-300 nm subcellular organelles that meet the rotational symmetry requirement, such as nuclear pore complex, primary cilium, and microtubule. In this Article, we will provide comprehensive analyses of this method by using experimental data and computational simulations. Finally, open source code of the 2D to 3D transformation algorithm (MATLAB) and simulations (Python) have also been developed.

Nuclear Transport and Accumulation of Smad Proteins Studied by Single-Molecule Microscopy.

Nuclear translocation of stimulated Smad heterocomplexes is a critical step in the signal transduction of transforming growth factor ß (TGF-ß) from transmembrane receptors into the nucleus. Specifically, normal nuclear accumulation of Smad2/Smad4 heterocomplexes induced by TGF-ß1 is involved in carcinogenesis. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-ß1 remains obscure. By combining a high-speed single-molecule tracking microscopy and Förster resonance energy transfer technique, we tracked the entire TGF-ß1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complexes in live cells, with a high single-molecule localization precision of 2 ms and <20 nm. Our single-molecule Förster resonance energy transfer data have revealed that in TGF-ß1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, we found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-ß1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-ß1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, we believe that the determined TGF-ß1-dependent transport configurations and efficiencies for the basal-state Smad or stimulated Smad heterocomplexes elucidate the basic molecular mechanism to understand their nuclear transport and accumulation.

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium.

Transport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α-tubulin utilizes diffusion besides IFT.

Single-molecule dynamics of site-specific labeled transforming growth factor type II receptors on living cells.

We achieved single-molecule imaging and tracking of the transforming growth factor type II receptor (TßRII) that was labeled by an organic dye via a genetically encoded unnatural amino acid (UAA) and the copper-free click chemistry. The stoichiometry, mobility and dimerization kinetics of individual TßRII molecules were determined.

Visualization of the post-Golgi vesicle-mediated transportation of TGF-ß receptor II by quasi-TIRFM.

Transforming growth factor ß receptor II (Tß RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live-cell fluorescence imaging techniques, in particular quasi-total internal reflection fluorescence microscopy, to imaging fluorescent protein-tagged Tß RII and monitoring its secretion process. We observed punctuate-like Tß RII-containing post-Golgi vesicles formed in MCF7 cells. Single-particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 µm/s. When stimulated by TGF-ß ligand, these receptor-containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post-Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tß RII-containing vesicles provide new information on intracellular Tß RII transportation. It also renders Tß RII a good model system for studying post-Golgi vesicle-trafficking and protein transportation.

Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization.

Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH4(+) uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane.

Single-molecule monitoring in living cells by use of fluorescence microscopy.

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.

A fluorescence aptasensor based on DNA charge transport for sensitive protein detection in serum.

A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.

Intercellular transportation of quantum dots mediated by membrane nanotubes.

In this work, we reported that the quantum dot (QD) nanoparticles could be actively transported in the membrane nanotubes between cardiac myocytes. Single particle imaging and tracking of QDs revealed that most QDs moved in a bidirectional mode along the membrane nanotubes with a mean velocity of 1.23 mum/s. The results suggested that QDs moving in the nanotubes were coordinately motivated by molecular motors. It provides new information for the study of intercellular transportation of nanoparticles.