Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study

All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapα2, kapβ1, kapβ1ΔN44, and kapβ2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy with a time resolution of 5 ms, achieving a colocalization precision of 30 nm. These measurements allowed defining the interaction sites with the NPCs with an unprecedented precision, and the comparison of the interaction kinetics with previous in vitro measurements revealed new insights into the translocation mechanism

A perspective of the dynamic structure of the nucleus explored at the single-molecule level

Cellular life can be described as a dynamic equilibrium of a highly complex network of interacting molecules. For this reason, it is no longer sufficient to “only” know the identity of the participants in a cellular process, but questions such as where, when, and for how long also have to be addressed to understand the mechanism being investigated. Additionally, ensemble measurements may not sufficiently describe individual steps of molecular mobility, spatial-temporal resolution, kinetic parameters, and geographical mapping. It is vital to investigate where individual steps exactly occur to enhance our understanding of the living cell. The nucleus, home too many highly complex multi-order processes, such as replication, transcription, splicing, etc., provides a complicated, heterogeneous landscape. Its dynamics were studied to a new level of detail by fluorescence correlation spectroscopy (FCS). Single-molecule tracking, while still in its infancy in cell biology, is becoming a more and more attractive method to deduce key elements of this organelle. Here we discuss the potential of tracking single RNAs and proteins in the nucleus. Their dynamics, localization, and interaction rates will be vital to our understanding of cellular life. To demonstrate this, we provide a review of the HIV life cycle, which is an extremely elegant balance of nuclear and cytoplasmic functions and provides an opportunity to study mechanisms deeply integrated within the structure of the nucleus. In summary, we aim to present a specific, dynamic view of nuclear cellular life based on single molecule and FCS data and provide a prospective for the future

A 4D view on mRNA

Imaging single molecules in live cells in 4+ D (space, time and colors) is crucial for studying various biological processes, especially for observing the behavior of RNA molecules within the nuclear landscape [1]. RNA molecules are known to serve a multitude of tasks such as being templates for protein translation or to act as enzymes for regulating countless reactions in the nucleus [1]. Studying RNA kinetics in living cells can provide new information on RNA function or even human diseases, for instance caused by viruses such as the human immunodeficiency virus (HIV) [2]. A challenge to imaging nuclear RNA function is that the nucleus as a whole undergoes major reformation during the cell cycle [1] but the time required to step through the sample limits the capability to image large numbers of rapidly moving particles in a 3D space

Fluorescence polarization control for on-off switching of single molecules at cryogenic temperatures [preprint]

Light microscopy allowing sub-diffraction limited resolution has been among the fastest developing techniques at the interface of biology, chemistry and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultra resolution structures, brings highly specific labeling of molecules in a large assemble to the table and inherently allows the detection of multiple colors, which enable the interrogation of multiple molecular species at the same time in the same sample. Here we discuss the problems to be solved in the coming years to aim for higher resolution and describe what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples like whole cells

An Automated Bayesian Pipeline for Rapid Analysis of Single-Molecule Binding Data [preprint]

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the protein\u27s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites

Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs [preprint]

Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 μm thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues

Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs [preprint]

Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 μm thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues

CRISPR-Cas9 nuclear dynamics and target recognition in living cells

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from \u3e3 h to as low as time

Nuclear transport of single molecules: dwell times at the nuclear pore complex

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs