Cellular Biology I: The Nucleus and DNA

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Life of Cells, Tissues and Microbes - Cellular Biology I

• Course topic is Cellular Biology I.
• Course presented by Lorena Di Pietro.
• Contact email is [email protected]

The Nucleus and DNA Organization

• This section focuses on the nucleus and its organization.
• Images of microscopic views of cells and DNA are part of the presentation.

The Nucleus

• The nucleus is the information processing and administrative center of the cell.
• It stores the cell's hereditary material (DNA).
• It coordinates various cell processes, including cell growth, intermediary metabolism, gene expression, protein synthesis, and reproduction.

The Nucleus (Further details)

• The nucleus is the largest cellular organelle.
• It's visible under a light microscope.
• Images of bright field microscopy of dermal fibroblasts and confocal microscopy of mesenchymal stromal cells are in the presentation.

The Nucleus (Eukaryotic Cells)

• All eukaryotic cells have a nucleus.
• Typically, a single nucleus is present per cell.
• The nucleus is usually rounded.
• Exceptions exist (e.g., megakaryocytes, erythrocytes, osteoclasts, and striated muscle cells).

The Nuclear Envelope

• The nuclear envelope separates the nucleus (nucleoplasm) from the cytoplasm.
• It is a double-layered membrane.
• It's permeable to certain molecules through nuclear pores.
• It connects to the endoplasmic reticulum.
• It plays a role in anchoring the nucleus to the cytoskeleton.

Nuclear Pore Complexes

• Nuclear pore complexes are gateways through the nuclear envelope.
• They facilitate the bidirectional movement of RNA and proteins.
• The number of complexes per nucleus varies widely (approximately 3,000-4,000).
• Each complex is comprised of approximately 30 different proteins (nucleoporins).
• The proteins are arranged in a repeating pattern with 8-fold rotational symmetry around the pore's opening, forming complex structures.
• Nucleoporins assemble to form a transmembrane ring, cytoplasmic ring, 8 cytoplasmic filaments, central scaffold, nuclear ring, and nuclear basket.
• The nuclear pores are not empty; they are filled with unstructured protein regions containing phenylalanine-glycine motifs (FG). The FG regions restrict the diffusion of large macromolecules, allowing smaller molecules to pass through.

Trafficking Across the NPC

• Proteins capable of traversing the nuclear pore complex (NPC) interact with nuclear transport receptors (importins and exportins).
• Importins transport macromolecules into the nucleus.
• Exportins transport macromolecules out of the nucleus.

Nuclear Import of Proteins

• A nuclear localization signal (NLS) in a protein guides import into the nucleus.
• The NLS binds to importin, a transport receptor.
• The importin–NLS complex interacts with cytoplasmic fibers of the nuclear pore complex and translocates through the channels.
• The GTP-binding protein Ran facilitates the release of the transported protein inside the nucleoplasm.
• Ran-GTP complex travels backward in the cytoplasm.
• Ran-GTP is hydrolyzed to Ran-GDP, releasing the cargo.
• Ran-GDP then returns to the nucleus where it is re-converted to GTP.

Nuclear Export of Proteins

• A nuclear export signal (NES) directs proteins out of the nucleus.
• The NES binds to exportin, a transport receptor.
• Exportin-NES complex translocates through the nuclear pore.
• GTP hydrolysis releases the cargo into the cytoplasm.
• Ran-GDP returns to the nucleus where it is recycled back to Ran-GTP.

Transport of RNAs

• Different types of RNA (mRNA, rRNA, snoRNA, miRNA, and tRNA) are assembled into ribonucleoproteins that travel across the nucleus.
• RNA molecules bind to specific NES proteins to move through the nucleus and to the cytoplasm, and only mature RNA molecules exit the nucleus.

The Nuclear Lamina

• The inner nuclear membrane is attached to a network of filaments, forming the nuclear lamina.
• The network is composed of intermediate filament proteins (lamins).
• The nuclear lamina plays a role in maintaining the shape and stability of the nucleus.
• It is involved in chromatin organization, interphase chromosome organization, mitosis, DNA replication and repair, transcription, gene expression, epigenetic modifications, cell proliferation, and differentiation.

The Nuclear Lamina: Mechanisms of Assembly and Disassembly

• Lamins are synthesized in the RER, and then translocate to the nucleus. They assemble into homodimers which then assemble into tetramers.
• The formation of these tetramers leads to fibers.
• The presentation classifies the four distinct lamin types (A, B1, B2, C).
• Specific sequence motifs or signals direct newly synthesized lamins to the envelope.
• Assembly and disassembly are regulated by phosphorylation.

The Nuclear Lamina in Diseases

• Laminopathies are genetic disorders caused by mutations in genes encoding proteins of the nuclear lamina.
• Pleiotropic roles of lamins contribute to the severe consequences of mutations.
• Certain laminopathies mainly involve heart and muscle, bone, fat, skin, growth and face abnormalities.
• Mutations in the lamin A gene (LMNA), leading to progerin, cause Hutchinson-Gilford Progeria Syndrome (HGPS).

The Nuclear Envelope: LINC Complex

• The LINC (linker of nucleoskeleton and cytoskeleton) complex is a network of proteins that links the nuclear envelope to the cytoskeleton outside the nucleus and to the nuclear lamina inside the nucleus.
• More than 80 proteins are part of this structure.

The Nuclear Envelope: SUN-KASH Protein Complexes

• SUN-KASH protein complexes establish connections between the cytoskeleton and the nucleoskeleton.
• SUN proteins connect the inner nuclear membrane to the nuclear lamina.
• KASH proteins connect the outer nuclear membrane to the nesprin proteins, which are linked to the cytoskeleton.

The Nucleolus

• The nucleolus is a large structure within a cell nucleus that is not bound by a membrane.
• It's a ribosome-producing factory.
• It's involved in synthesizing and processing rRNA.
• It is the site of ribosome assembly.

Ribosomes

• Ribosomes are the cellular machinery for protein translation.
• Eukaryotic cells contain large numbers of ribosomes.
• Eukaryotic ribosomes include four types of rRNA: 5S, 5.8S, 18S, and 28S.
• Ribosomes use tRNA to assemble amino acids into proteins using mRNA as instructions.

The Nucleolus (Components)

• The nucleolus has three main components:
• Fibrillar Center (FC): houses the rDNA for rRNA transcription.
• Dense Fibrillar Component (DFC): contains proteins involved in rRNA processing.
• Granular Component (GC): contains proteins involved in ribosome biogenesis.

The Nucleolus (Multiple Functions)

• The nucleolus participates in several cell processes, including regulation of the cell cycle through sequestration, processing some tRNAs and snRNAs, maturation, sensing cell stress, replication, gene expression, epigenetic modifications, and cell proliferation and differentiation.
• The nucleolus is important for multiple processes, including some aspects of viral replication and regulation of cellular activities.

Other Components of the Nucleoplasm

• Cajal bodies (coiled bodies) and interchromatin granule clusters are other structural components of the nucleoplasm.
• These structures are involved in RNA processing and are important for various cellular processes.
• These are dynamic and their activity depends on the needs of the cell. Their high concentration ensures that involved processes are rapid and efficient.

DNA Organization

• DNA, in eukaryotic cells, is organized in chromosomes.
• Each chromosome contains a single, linear DNA molecule.
• Chromatin is the packaged form of DNA associated with proteins.
• The structure of chromatin changes based on the functional state and activity of the cell.
• In eukaryotes, DNA is organized in chromosomes.
• In prokaryotes, DNA is usually a single, circular molecule in a region called the nucleoid.
• Prokaryotes may also have smaller circular DNA molecules called plasmids.
• Plasmids allow bacteria to cope with stressful conditions like antibiotic resistance.

DNA vs. RNA

• DNA is a double-stranded molecule, while RNA is usually single-stranded.
• DNA uses the bases adenine, guanine, cytosine, and thymine.
• RNA uses the bases adenine, guanine, cytosine, and uracil.
• DNA is usually a long string in cells, packaged into compact structures using proteins (histones), while RNA commonly performs other functions in the cells.

DNA Organization - Further details

• In eukaryotic cells, the chromosomes are composed of DNA and associated proteins.
• DNA and histone proteins are packaged into nucleosomes.
• Nucleosomes form a 10 nm fiber to which another level of packing can be added, forming a 30 nm chromatin fiber.
• The fibers form loops attached to a protein scaffold.
• The 30 nm fibers and protein scaffold are further condensed into the mitotic chromosome structures.
• The chromatin is organized differently based on the level of activity of the DNA.
• The organization of chromosomes is essential for regulating DNA access to enzymes and regulatory proteins.
• The number of chromosomes varies among species and is not related to the species’ biological complexity.

Heterochromatin/Euchromatin

• Heterochromatin is a condensed form of chromatin. It's typically located in the periphery of the nucleus.
• Euchromatin is a dispersed form of chromatin, which is generally more active in transcription.
• Constitutive heterochromatin is permanently inactive and includes pericentromeric and telomeric regions.
• Facultative heterochromatin involves DNA regions that are selectively inactive in some cell types. An example of this is X chromosome inactivation in females leading to the formation of Barr bodies inside cells.
• The positioning of particular genes can influence their expression. Incorrect heterochromatinization can lead to diseases, (example: b-globin gene).

Chromosome Structure

• A chromosome is composed of two identical chromatids, joined together at the centromere.
• The short arm is labeled 'p' and the long arm is labeled 'q.'
• The ends of each arm are called telomeres.