NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure Freezing/Freeze Substitution of Yeast, Worms, and Plants.

The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle.

Scanning Electron Microscopy (SEM) and Immuno-SEM of Nuclear Pore Complexes from Amphibian Oocytes, Mammalian Cell Cultures, Yeast, and Plants.

Scanning electron microscopy (SEM) can be used to image nuclear pore complex (NPC) surface structure of from a number of organisms and model systems. With a field emission SEM , this is a medium resolution technique where details of the organization of various components can be directly imaged. Some components, such as the NPC baskets and cytoplasmic filaments, are difficult to visualize in any other way.

Nucleoporin TPR Affects C2C12 Myogenic Differentiation via Regulation of Expression.

The nuclear pore complex (NPC) has emerged as a hub for the transcriptional regulation of a subset of genes, and this type of regulation plays an important role during differentiation. Nucleoporin TPR forms the nuclear basket of the NPC and is crucial for the enrichment of open chromatin around NPCs. TPR has been implicated in the regulation of transcription; however, the role of TPR in gene expression and cell differentiation has not been described.

Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery.

The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy.

Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution.

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1.

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data.

The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data.

Immunogold Labeling for Scanning Electron Microscopy.

Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components, and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo, or real 3D at magnifications ranging from about 10× to 1,000,000×. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualized.

Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast Saccharomyces cerevisiae.

Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility.

Arp2/3 complex subunit ARPC2 binds to microtubules.

Arp2/3 complex plays a fundamental role in the nucleation of actin filaments (AFs) in yeasts, plants, and animals. In plants, the aberrant shaping and elongation of several types of epidermal cells observed in Arp2/3 complex knockout plant mutants suggest the importance of Arp2/3-mediated actin nucleation for various morphogenetic processes. Here we show that ARPC2, a core Arp2/3 complex subunit, interacts with both actin filaments (AFs) and microtubules (MTs).

Paxillin-dependent regulation of IGF2 and H19 gene cluster expression.

Paxillin (PXN) is a focal adhesion protein that has been implicated in signal transduction from the extracellular matrix. Recently, it has been shown to shuttle between the cytoplasm and the nucleus. When inside the nucleus, paxillin promotes cell proliferation.

Imaging yeast NPCs: from classical electron microscopy to Immuno-SEM.

Electron microscopy (EM) has been used extensively for the study of nuclear transport as well as the structure of the nuclear pore complex (NPC) and nuclear envelope. However, there are specific challenges faced when carrying out EM in one of the main model organisms used: the yeast, Saccharomyces cerevisiae. These are due to the presence of a cell wall, vacuoles, and a densely packed cytoplasm which, for transmission EM (TEM), make fixation, embedding, and imaging difficult.

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains.

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic movement. The central channel contains proteins with phenylalanine-glycine (FG) repeats, or variations (GLFG, glycine-leucine-phenylalanine-glycine). These are 'intrinsically disordered' and often represent weak interaction sites that become ordered upon interaction.

Imaging plant nuclei and membrane-associated cytoskeleton by field emission scanning electron microscopy.

Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes.

Multiple actin isotypes in plants: diverse genes for diverse roles?

Plant actins are encoded by a gene family. Despite the crucial significance of the actin cytoskeleton for plant structure and function, the importance of individual actin isotypes and their specific roles in various plant tissues or even single cells is rather poorly understood. This review summarizes our current knowledge about the plant actin gene family including its evolution, gene and protein structure, and the expression profiles and regulation.

Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex.

Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing.

Immunogold labelling for scanning electron microscopy.

Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo or real 3D at magnifications of anything from about x10 to x1,000,000. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualised.

Immunoelectron microscopy of cryofixed freeze-substituted Saccharomyces cerevisiae.

Immunolabelling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an ideal way to fix cellular structure. However, its use for immunolabelling has remained limited because of the low frequency of labelling due to loss of protein antigenicity or accessibility.

Relationships at the nuclear envelope: lamins and nuclear pore complexes in animals and plants.

The nuclear envelope comprises a distinct compartment at the nuclear periphery that provides a platform for communication between the nucleus and cytoplasm. Signal transfer can proceed by multiple means. Primarily, this is by nucleocytoplasmic trafficking facilitated by NPCs (nuclear pore complexes).

The role of actin isoforms in somatic embryogenesis in Norway spruce.

Background: Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs.

Results: Application of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types.

Nucleocytoplasmic transport in yeast: a few roles for many actors.

Eukaryotic cells have developed a series of highly controlled processes of transport between the nucleus and cytoplasm. The present review focuses on the latest advances in our understanding of nucleocytoplasmic exchange of molecules in yeast, a widely studied model organism in the field. It concentrates on the role of individual proteins such as nucleoporins and karyopherins in the translocation process and relates this to how the organization of the nuclear pore complex effectively facilitates the bidirectional transport between the two compartments.

Nuclear envelope and nuclear pore complex structure and organization in tobacco BY-2 cells.

The nuclear envelope (NE) is a fundamental structure of eukaryotic cells with a dual role: it separates two distinct compartments, and enables communication between them via nuclear pore complexes (NPCs). Little is known about NPCs and NE structural organization in plants. We investigated the structure of NPCs from both sides of the NE in tobacco BY-2 cells.

Tobacco Arp3 is localized to actin-nucleating sites in vivo.

The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L.

The C-terminal variable region specifies the dynamic properties of Arabidopsis microtubule-associated protein MAP65 isotypes.

The microtubule-associated protein, MAP65, is a member of a family of divergent microtubule-associated proteins from different organisms generally involved in maintaining the integrity of the central spindle in mitosis. The dicotyledon Arabidopsis thaliana and the monocotyledon rice (Oryza sativa) genomes contain 9 and 11 MAP65 genes, respectively. In this work, we show that the majority of these proteins fall into five phylogenetic clades, with the greatest variation between clades being in the C-terminal random coil domain.

A new model for nuclear lamina organization.

Lamins are intermediate filament proteins that form a network lining the inner nuclear membrane. They provide mechanical strength to the nuclear envelope, but also appear to have many other functions as reflected in the array of diseases caused by lamin mutations. Unlike other intermediate filament proteins, they do not self-assemble into 10 nm filaments in vitro and their in vivo organization is uncertain.

ARP2 and ARP3 are localized to sites of actin filament nucleation in tobacco BY-2 cells.

Complete depolymerization of actin filaments (AFs) at low temperature (0 degrees C) is followed by the formation of transient actin structures at 25 degrees C in tobacco BY-2 cells (Nicotiana tabacum L.). Using antibodies against fission yeast actin-related proteins (ARP2 and ARP3), we show here that transient actin structures (dots, dotted filaments, rods) colocalize with epitopes stained by these antibodies and thus are likely to represent sites of actin filament nucleation (SANs).