Chapter 9: Nuclear Structure and Transport

9.1 Introduction

The nucleus contains most of the cell's DNA, allowing for sophisticated regulation of gene expression.
The nuclear envelope is a double membrane that surrounds the nucleus.
The nucleus contains subcompartments that are not membrane-bounded.
The nuclear envelope contains pores used for transporting RNAs, proteins, and small molecules between the nucleus and the cytoplasm.
Eukaryotic cells are defined by the presence of a "true nucleus".
The nucleus acts as a center for controlling cellular activities.
Antony van Leeuwenhoek may have been the first to observe nuclei.
Felice Fontana is credited with the actual discovery of the nucleus.
Robert Brown was the first to call these structures nuclei, a term derived from the Latin for kernel.
The nuclear envelope encloses the nucleus and consists of two membranes separated by a lumen contiguous with the endoplasmic reticulum (ER).
Nuclear pore complexes (NPCs) span the nuclear envelope and are the channels through which macromolecules pass between the nucleus and cytoplasm.
Proteins passing through NPCs do not generally need to be unfolded for transport.
The nucleolus is a nuclear subcompartment where ribosomal RNAs (rRNAs) are synthesized and ribosomal subunits are assembled.
Other subcompartments, like speckles and Cajal bodies, are revealed by immunofluorescence microscopy.

9.2 Nuclei vary in appearance according to cell type and organism

Nuclei range in size from about 1 $μm$ to more than 10 $μm$ in diameter.
Most cells have a single nucleus, but some cells contain multiple nuclei, and a few cell types lack nuclei.
The percentage of the genome that is heterochromatin varies among cells and increases as cells become more differentiated.
The size of the nucleus is related to the amount of DNA it contains.
Smallest nuclei (approximately 1 $μm$ ) are found in single-celled eukaryotes like Saccharomyces cerevisiae.
Nuclei of cells of many multicellular organisms are 5-10 $μm$ in diameter.
Oocytes of the frog Xenopus laevis have nuclei up to 1 mm in diameter and are widely used in cell biologic and biochemical studies.
In most cells, the nucleus is spherical or oblong to minimize surface area.
The percentage of total cell volume occupied by nuclei varies:
- 1%-2% in yeast cells
- 10% in most somatic cells
- 40%-60% in cells with less need for cytoplasmic functions.
Multinucleate cells can form when a cell undergoes nuclear division (karyokinesis) without cell division (cytokinesis).
- Early embryos of Drosophila melanogaster contain hundreds of nuclei within a common cytoplasm.
Multinucleate cells also form when cells fuse to form a syncytium.
- Mature muscle cells (myocytes) form by fusion of precursor cells (myoblasts).
A few differentiated cell types lack nuclei:
- Mammalian mature red blood cells
- Blood platelets
- Some cells in the lens of the vertebrate eye.
The shape and appearance of the nucleus allow different types of cells to be distinguished from one another.
Leukemias are blood diseases where white blood cells are produced in much greater numbers than normal, and the morphology of their nuclei is used in diagnosis.
The fraction of the genome that is heterochromatin is another nuclear feature that helps to identify some cell types.
As immature erythroblasts differentiate into mature erythrocytes, more and more of the DNA becomes heterochromatin.
The increase in heterochromatin correlates with the permanent silencing of most genes.
In some species, including humans, the erythrocyte nucleus is ultimately ejected.
Red blood cells acquire their biconcave disk shape only after the nucleus is lost, which is critical for their easy passage through tiny capillaries.

9.3 Chromosomes occupy distinct territories

Nuclei are highly organized and contain many subcompartments, even though they lack internal membranes.
Each chromosome occupies a distinct region or territory, which prevents chromosomes from becoming entangled with one another.
The nucleus contains both chromosome domains and interchromosomal regions.
Chromosomes do not intertwine but are spatially ordered, with each in a separate chromosome region, domain, or territory (approximately 2 $μm$ in diameter).
Isolating each chromosome in its own territory avoids tangling and breakage during mitosis.
Telomeres are anchored to the nuclear envelope in many cell types, likely helping to prevent tangling of chromosomes.
Specific chromosomes tend to cluster nearer to some chromosomes than to others.
Different arrangements of chromosome territories exist in different cell types and tissues.
Colocalization of coordinately regulated genes on different chromosomes can occur by clustering and forming a transcription factory.
Chromatin does not fill the entire nucleus.
There are chromosome domains (regions where chromatin is located) and adjacent chromatin-free regions called interchromosomal domains.
Interchromosomal domains contain poly(A) RNAs undergoing final processing and diffusing to the nuclear periphery for export.
Highly transcribed genes tend to be located at the periphery of chromatin domains adjacent to interchromosomal domains.
The genes adjacent to interchromosomal regions vary according to cell type, as does the organization of chromosomes within their territories.
Location of highly transcribed genes adjacent to interchromosomal domains facilitates mRNA diffusion to NPCs for export.
Actively transcribed genes are sometimes located near NPCs, which may also enhance efficient mRNA export.

9.4 The nucleus contains subcompartments that are not membrane-bounded

Nuclear subcompartments are not membrane-bounded.
rRNA is synthesized and ribosomal subunits are assembled in the nucleolus.
Genes that encode rRNAs are present on multiple chromosomes that cluster together to form nucleolar subcompartments.
mRNA splicing factors are stored in nuclear speckles and move to sites of transcription where they function.
Other nuclear bodies have been identified using antibodies; some bodies may concentrate specific nuclear proteins, but the functions of most are unknown.
Key events in the nucleus include gene expression steps, such as transcription and RNA processing, which occur at discrete locations.
The most prominent subcompartment of the nucleus is the nucleolus, where rRNAs are synthesized and processed, and ribosomal subunits are assembled.
Most normal cells have a single nucleolus, but multiple nucleoli can be found in some cells.
The size of the nucleolus varies, depending on the amount of ribosome biogenesis in a given cell.
The nucleolus contains all components needed for ribosomal subunit assembly, including rRNA genes, rRNAs, synthesis and processing enzymes, and ribosomal proteins.
Transcription of rRNA genes, rRNA processing, and subunit assembly take place in different regions within the nucleolus.
Although the nucleolus lacks a membrane, most of its proteins and RNAs are found only there.
The nucleolus is detectable only when ribosomal subunit production is occurring, and it disappears when rRNA transcription is prevented.
The nucleolus represents the coming together of rRNA genes, transcription factors, and RNA polymerase I molecules.
Newly synthesized rRNAs attract ribosomal proteins and processing factors to assemble ribosomal subunits.
The nucleolus is disassembled during mitosis and reassembled when mitosis has been completed, possibly due to inhibition of transcription.
The nucleolus is also involved in tRNA transcription and processing.
Proteins that do not participate in ribosomal subunit formation are also found in the nucleolus.
Some cell cycle-regulated proteins may be sequestered in the nucleolus until required.
Proteomic analyses indicate nucleoli contain more than 400 different polypeptides.
Several smaller, discrete subcompartments of the nucleus, called nuclear bodies, have been detected:
- Speckles
- Cajal bodies
- Gemini bodies
- Promyelocytic leukemia (PML) bodies
One function of nuclear bodies may be to increase the efficiency of biologic processes by concentrating multiple macromolecules.
RNA splicing factors are spatially organized in the nucleus in speckles, with approximately 20-50 speckles per cell.
Speckles store splicing factors rather than being splicing factories, as they do not contain pre-mRNA.
Splicing is believed to occur in diffusely stained areas where polyadenylated RNA and splicing factors are detected.
If transcription is blocked, splicing factors relocate to speckles.
Cajal bodies contain coilin and are believed to be sites where snRNAs and small nucleolar RNAs are modified post-transcriptionally and assembled into ribonucleoprotein complexes.
Gemini bodies are not found in all cells, and some components are also found in Cajal bodies.
PML bodies contain a protein related to one first found in patients with PML.
The precise function of PML bodies is unknown, but they may be storage sites for transcription factors and chromatin-modifying enzymes.
Many of these smaller nuclear bodies appear to be absent from the small nuclei of unicellular eukaryotes such as yeast.
Yeast cells do contain nuclear bodies similar to Cajal bodies.

9.5 Some processes occur at distinct nuclear sites and may reflect an underlying structure

The nucleus contains replication sites where DNA is synthesized.
The nucleus may contain a nucleoskeleton that could help to organize nuclear functions.
Macromolecular machinery for DNA replication and RNA splicing may be connected to an underlying nuclear structure.
During the early part of S phase, cells contain many sites of DNA replication that coalesce into a few dozen larger sites called replication factories or foci as S phase proceeds.
Each replication factory contains dozens or hundreds of origins.
These foci are distinct from various nuclear bodies, with some disappearing and new ones forming throughout S phase.
Transcription may also occur in a limited number of sites called transcription factories.
Localization of nuclear processes to discrete sites suggests the possibility of an underlying structure in the nucleus.
The nuclear lamina is highly organized and constitutes part of a nucleoskeleton.
Some studies suggest a filamentous network, called the nuclear matrix, may exist in the nucleoplasm.
The nuclear matrix is seen only if nuclei are treated with detergents, DNase, and high salt concentration, extracting much material and leaving only insoluble proteins and some RNA.
RNA appears to play a key organizing role in the nuclear matrix.
Some cell biologists believe the nuclear matrix is an artifact because it can only be seen after harsh extraction.
An underlying organization of some sort is likely, given the complexity and accuracy required for nuclear processes.
One possible function for an underlying nuclear structure is the organization of the machinery for replication, transcription, and RNA processing.
Replisomes, RNA polymerase II holoenzyme complexes, and spliceosomes may be attached to an underlying structure.
When replication, transcription, and splicing occur, the protein machineries may be fixed, and the nucleic acids may move through the complexes.
Replication factories show little mobility, in contrast with speckles and other nuclear bodies.

9.6 The nucleus is bounded by the nuclear envelope

The nucleus is surrounded by a nuclear envelope consisting of two complete membranes.
The outer nuclear membrane is continuous with the membranes of the ER, and the lumen of the nuclear envelope is continuous with the lumen of the ER.
The nuclear envelope contains numerous NPCs, the only channels for transport of molecules and macromolecules between the nucleus and the cytoplasm.
The nuclear envelope consists of two concentric membranes: the outer nuclear membrane (ONM) and the inner nuclear membrane (INM).
Each nuclear membrane contains a complete phospholipid bilayer, some common proteins, and some proteins unique to it.
Except in some single-celled eukaryotes, a network of filaments, woven into a meshwork structure, supports the inner nuclear membrane (the nuclear lamina).
The outer nuclear membrane is continuous with the membranes of the ER and is covered with ribosomes engaged in protein synthesis.
The space between the outer and inner nuclear membranes is the lumen of the nuclear envelope, which is continuous with the lumen of the ER.
Each of the two membranes is 7-8 nm thick, and the nuclear envelope lumen is 20-40 nm wide.
The most conspicuous features of the nuclear envelope are the NPCs that serve as channels for movement between the cytoplasm and nucleus.
The number of NPCs per cell varies and is correlated with the level of nuclear transport required.
Many mammalian cell types contain about 3,000-4,000 NPCs.
Yeast cells contain 150-250 NPCs, while Xenopus oocytes contain several million.
Xenopus oocytes are favorites system to study NPC structure, composition, and function.
The double membrane of the nucleus shares this property with mitochondria and chloroplasts.
The endosymbiont hypothesis proposes that these organelles arose during evolution when cells endocytosed other cells.
The ingested cell would then be surrounded by two membranes: its own and that of the engulfing cell.

9.7 The nuclear lamina underlies the nuclear envelope

The nuclear lamina is constructed of intermediate filament proteins called lamins.
The nuclear lamina is located beneath the inner nuclear membrane and is physically connected to it by lamina-associated integral membrane proteins.
The nuclear lamina plays a role in nuclear envelope assembly and may provide physical support for the nuclear envelope.
Proteins connect the nuclear lamina to chromatin; this may allow the nuclear lamina to organize DNA replication and transcription.
Protein complexes that interact with the nuclear lamina cross the nuclear envelope and link the cytoskeleton to the nuclear interior.
Yeast and some other unicellular eukaryotes lack a nuclear lamina.
A common feature of the nuclei of metazoan organisms is the presence of the nuclear lamina, an intermediate filament meshwork lying just beneath the inner nuclear membrane.
Nuclear lamina proteins are related to the keratin proteins of cytoplasmic intermediate filaments.
Both lamin proteins and keratins are called intermediate filament proteins because the size of the filaments they form (10-20 nm in diameter) is intermediate between that of actin microfilaments (7 nm in diameter) and microtubules (25 nm in diameter).
The nuclear lamina is interrupted by NPCs, which are anchored to the nuclear lamina.
In addition to the lamins, the nuclear lamina also contains a set of integral membrane proteins called lamina-associated proteins (LAPs), some of which mediate interactions between the lamina and the inner nuclear membrane.
The lamina is anchored to the inner nuclear membrane by two types of interactions:
- Between lamin proteins and integral membrane proteins of the inner nuclear membrane.
- By attachment of the lamin polypeptide chain to a farnesyl lipid group in the inner nuclear membrane.
SUN (Sadlp, UNC-84) domain proteins interact with proteins of the outer nuclear membrane called KASH (Klarsicht, ANC-1, Syne Homolgy) proteins, forming a physical linkage that spans the nuclear envelope and couples the cytoskeleton to the nuclear interior.
This linkage complex is called the LINC complex or the linker of nucleoskeleton and cytoskeleton complex and plays an essential role in transmission of forces generated by the cytoskeleton to the nucleus.
Plant genomes do not encode lamins, but plants appear to contain other structural proteins that function like the lamins of animal cells.
Yeast (such as S. cerevisiae and Schizosaccharomyces pombe) and other unicellular eukaryotes appear to lack lamins and a lamina.
Yeast cells undergo a closed mitosis, where the nuclear envelope remains intact at all times, while cells of multicellular eukaryotes disassemble the nuclear envelope early in mitosis.
The lamina is believed to play a central role in rebuilding nuclear organization at this time.
The lamina may provide essential structural support for the much larger nuclear envelope found in metazoan cells.
The nuclear lamina interacts with chromatin and may be needed for DNA replication to occur.
When sperm chromatin is added to X. laevis egg extracts, a nuclear envelope forms around the sperm chromatin.
These nuclei enlarge, and the chromosomes decondense.
If the lamins are removed from these extracts, nuclear envelopes still assemble around the sperm chromatin, but these nuclei are small and fragile, and DNA replication does not occur.
These results indicate that the lamina might be important for organizing the chromatin so it can be replicated.
Mutations affecting lamins and LAPS are associated with several genetic diseases, called laminopathies, primarily affecting muscle.
An altered nuclear lamina appears to make the nucleus more fragile and more susceptible to injury.

9.8 Large molecules are actively transported between the nucleus and cytoplasm

Uncharged molecules smaller than 100 Da can pass through the membranes of the nuclear envelope.
Molecules and macromolecules larger than 100 Da cross the nuclear envelope by moving through NPCs.
Particles up to 9 nm in diameter (corresponding to globular proteins up to 40 kDa) can pass through NPCs by passive diffusion, as can metabolites, nucleotides, and other small molecules.
Larger macromolecules are actively transported through NPCs and must contain specific information to be transported.
Uncharged molecules smaller than 100 Da, including water, can diffuse freely through phospholipid bilayers, but other molecules and macromolecules that are transported across the nuclear envelope move through NPCs.
The process of moving through the NPC is called translocation.
The movement of molecules (>100 Da) and macromolecules between the nucleus and the cytoplasm can be studied by labeling molecules with radioactivity or fluorescent dyes and injecting them into large cells, such as amphibian oocytes.
Small molecules, such as glucose-6-phosphate or fluorescein, cross the nuclear envelope extremely rapidly, within several seconds.
At equilibrium, the concentration of these types of small molecules is the same on the two sides of the nuclear envelope.
Protein-assisted transport functions poorly or not at all at 4° C, whereas diffusion occurs at nearly equal rates at 4° C and at physiologic temperatures.
The maximum size of particles able to freely diffuse across the nuclear envelope was determined by injecting gold particles of precise sizes.
Particles 9 nm diameter or smaller can move into and out of the nucleus by passive diffusion through the nuclear pore, corresponding to a globular protein of approximately 40 kDa.
The rate of diffusion is the same in both directions.
Proteins with dimensions larger than about 9 nm cannot diffuse freely through nuclear pores; these proteins are actively and selectively transported.
Nuclear import and export of proteins are selective processes sensitive to ATP depletion and do not occur at 4° C, implying that transport is an energy-dependent process.

9.9 Nuclear pore complexes are symmetric channels

NPCs are symmetric structures found at sites where the inner and outer nuclear membrane are fused.
Each NPC in human cells has a mass of approximately 120 x 106 Da, which is 40 times that of a ribosome, and is constructed from multiple copies of about 30 proteins.
NPCs contain fibrils that extend into the cytoplasm and a basket-like structure that extends into the nucleus.
NPCs in human cells are estimated to have a molecular weight of approximately 112 x 106 Da and an outside diameter of about 120 nm.
NPCs are constructed from multiple copies of approximately 30 different polypeptides, the nucleoporins.
NPCs are barrel-like structures that span the nuclear envelope and extend somewhat beyond the planes of both its membranes, creating a structure in the form of an annulus or a ring.
Most NPCs have eightfold rotational symmetry.
The cytoplasmic and nuclear faces of the NPC look quite different.
The parts of the NPC that extend into the cytoplasm and nucleoplasm are called terminal structures.
The terminal structures emanating from the cytoplasmic face of the NPC are eight relatively short fibrils that extend about 100 nm into the cytoplasm.
On the nuclear face are similar fibrils joined to a ring, referred to as the nuclear basket, or "fish trap."
In some cells from multicellular organisms, additional fibrils extend from the basket structure deep into the interior of the nucleus.
The terminal structures on both the cytoplasmic and nuclear sides are the sites where molecules to be transported first interact with the NPC and, after translocation through the NPC channel, have their final interactions with the NPC.
Structural models of NPCs have been derived from the analysis of hundreds of high-resolution electron micrographs of individual NPCs.
The NPCs of S. cerevisiae and other unicellular eukaryotes have a mass of approximately 66 x 106 Da, approximately half the mass of NPCs from multicellular organisms.
In spite of the difference in size, the overall structure and most of the nucleoporins are conserved.
Metazoan and yeast NPCs do not differ in the size of the channels at the center of NPCs or in their transport properties.
Wherever NPCs are located, the inner and outer nuclear membranes are fused, and the NPC can be thought of as a membrane tunnel lined with proteins.
Fusion is an integral part of the process of assembling an NPC within the nuclear envelope.
NPCs are anchored in the nuclear envelope by integral membrane proteins that are part of the core structure.
These proteins extend into the lumen of the nuclear envelope.
NPCs penetrate the nuclear lamina and are also anchored to it.
The best model of the NPC indicates that it consists of multiple rings and spokes joined in an intricate manner.
In some cells, NPCs are found not only in the nuclear envelope but also in structures called annulate lamellae, which are cytoplasmically located stacks of double membranes containing NPCs.
Annulate lamellae are most commonly seen in oocytes of both invertebrates and vertebrates but have been observed in other types of cells as well.
Because they lack an underlying lamina, annulate lamellae have proven to be a valuable source of NPCs for biochemical and cytologic studies.
The NPCs in annulate lamellae appear to have the same structure and composition as NPCs in the nuclear envelope.

9.10 Nuclear pore complexes are constructed from nucleoporins

The proteins of NPCs are called nucleoporins.
Many nucleoporins contain repeats of short sequences such as Gly-Leu-Phe-Gly, X-Phe-X-Phe-Gly, and X-X-Phe-Gly, which are believed to interact with transport factors during transport.
Some nucleoporins are transmembrane proteins that anchor NPCs in the nuclear envelope.
All nucleoporins of yeast NPCs and most if not all nucleoporins of mammalian NPCs have been identified.
NPCs are disassembled and reassembled during mitosis in cells undergoing an open mitosis.
Some nucleoporins are dynamic: they rapidly associate with and dissociate from NPCs.
The proteins that make up NPCs are called nucleoporins.
Two major approaches contributed to identifying the complete set of yeast nucleoporins:
- A genetic approach to isolate mutants defective for nuclear transport.
- Biochemical purification of NPCs from isolated nuclear envelopes.
Solubilization of yeast NPCs was facilitated by yeast's lack of a nuclear lamina.
Yeast nucleoporins were separated by gel electrophoresis and identified using mass spectrometry.
Approximately 30 proteins have been identified as yeast nucleoporins.
Most nucleoporins from metazoans have also been identified.
Metazoan NPCs have approximately twice the mass of yeast NPCs, but the number of different nucleoporins is approximately the same.
Many nucleoporins in metazoan NPCs are orthologous to most or perhaps all yeast nucleoporins.
Vertebrate nuclear pore proteins have been identified using biochemical and immunologic approaches and RNA interference.
One characteristic of many nucleoporins is the presence of multiple repeats of short sequences, believed to interact with transport factors.