Dynamics of nuclear export of pre-ribosomal subunits revealed by high-speed single-molecule microscopy in live cells.

We present a study on the nuclear export efficiency and time of pre-ribosomal subunits in live mammalian cells, using high-speed single-molecule tracking and single-molecule fluorescence resonance energy transfer techniques. Our findings reveal that pre-ribosomal particles exhibit significantly higher nuclear export efficiency compared to other large cargos like mRNAs, with around two-thirds of interactions between the pre-60S or pre-40S and the nuclear pore complexes (NPCs) resulting in successful export to the cytoplasm. We also demonstrate that nuclear transport receptor (NTR) chromosomal maintenance 1 (CRM1) plays a crucial role in nuclear export efficiency, with pre-60S and pre-40S particle export efficiency decreasing by 11-17-fold when CRM1 is inhibited.

Telomere dysfunction implicates POT1 in patients with idiopathic pulmonary fibrosis.

Exonic sequencing identified a family with idiopathic pulmonary fibrosis (IPF) containing a previously unreported heterozygous mutation in POT1 p.(L259S). The family displays short telomeres and genetic anticipation.

POT1-TPP1 binding stabilizes POT1, promoting efficient telomere maintenance.

Telomeric POT1-TPP1 binding is critical to telomere maintenance and disruption of this complex may lead to cancer. Current data suggests a reduction of intracellular POT1 levels in the absence of TPP1. Here we provide evidence of POT1 plasticity that contributes to its lack of stability in the absence of TPP1 binding.

Pol α-primase dependent nuclear localization of the mammalian CST complex.

The human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by inhibiting telomerase access to the telomeric overhang and facilitates lagging strand fill in by recruiting DNA Polymerase alpha primase (Pol α-primase) to the telomeric C-strand. Here we reveal that CST has a dynamic intracellular localization that is cell cycle dependent.

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high-speed super-resolution microscopy.

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown.

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.

Nuclear export of mRNA molecules studied by SPEED microscopy.

The nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cell imaging allowing a more comprehensive understanding of the dynamics of mRNA export through native NPCs.

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex.

The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex.

SPEED Microscopy and Its Application in Nucleocytoplasmic Transport.

In eukaryotic cells, the nuclear pore complexes (NPCs) selectively mediate the bidirectional trafficking of macromolecules between the cytoplasm and the nucleus. The selective barrier is formed by intrinsically disordered phenylalanine-glycine (FG) nucleoporins anchored on the wall of the submicrometer NPC, which allows for passive diffusion and facilitated translocation through the nuclear pore. Dysfunction of nucleocytoplasmic transport has been associated with many human diseases.

Super-resolution imaging of nuclear import of adeno-associated virus in live cells.

Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level.

High-resolution imaging reveals new features of nuclear export of mRNA through the nuclear pore complexes.

The nuclear envelope (NE) of eukaryotic cells provides a physical barrier for messenger RNA (mRNA) and the associated proteins (mRNPs) traveling from sites of transcription in the nucleus to locations of translation processing in the cytoplasm. Nuclear pore complexes (NPCs) embedded in the NE serve as a dominant gateway for nuclear export of mRNA. However, the fundamental characterization of export dynamics of mRNPs through the NPC has been hindered by several technical limits.