celbiologie 04 deel 1 nucleus.pdf

# Structure and function of the nucleus The nucleus, a prominent organelle in eukaryotic cells, houses the cell's genetic material and controls its growth and reproduction, with its structure intricately linked to its diverse functions [5](#page=5). ### 1.1 Ultrastructure of the nucleus The nucleus, particularly during the interphase when the cell is not dividing, is characterized by several key components observable under an electron microscope [3](#page=3) [5](#page=5): #### 1.1.1 Nuclear envelope The nuclear envelope, also referred to as the nuclear membrane, is a double phospholipid membrane that encloses the nucleus. It is punctuated by nuclear pores, which regulate the passage of molecules between the nucleus and the cytoplasm. Ribosomes can be found attached to the outer surface of the nuclear envelope, similar to their presence on the rough endoplasmic reticulum [3](#page=3) [5](#page=5). #### 1.1.2 Nucleolus The nucleolus, or nucleoli in plural, is a dense structure found within the karyoplasm. Its primary function is the synthesis of ribosomes. The presence and distinctness of the nucleolus can be an indicator of the cell's metabolic state [3](#page=3) [5](#page=5). #### 1.1.3 Karyoplasm The karyoplasm, or nucleoplasm, is the fluid-filled matrix within the nuclear envelope. It serves as a compartment containing essential building blocks for DNA and RNA, as well as enzymes crucial for nuclear processes, such as DNA and RNA polymerases involved in transcription [3](#page=3) [5](#page=5). #### 1.1.4 Chromatin Chromatin represents the form of DNA and protein complex found within the nucleus during interphase. It appears as a granular structure under light microscopy. There are two main types of chromatin, distinguished by their degree of condensation and transcriptional activity [3](#page=3) [6](#page=6): * **Euchromatin:** This form is less condensed and contains the majority of the cell's active genes. It is metabolically active DNA [3](#page=3) [6](#page=6). * **Heterochromatin:** This form is highly condensed and generally associated with transcriptionally inactive regions of the genome. It is often found along the inner side of the nuclear envelope and around the nucleolus. A pycnotic nucleus is characterized by the presence of significant heterochromatin [3](#page=3) [6](#page=6). > **Tip:** The state of chromatin (euchromatin vs. heterochromatin) is directly correlated with the metabolic state of the cell [5](#page=5) [6](#page=6). During cell division, chromatin further condifies into a highly compact structure known as chromosomes [6](#page=6). ##### 1.1.4.1 Levels of chromatin compaction Chromatin exhibits sequential levels of compaction in eukaryotic cells [7](#page=7): 1. **Nucleosomes:** The basic structural unit, where DNA is wrapped around a core of histone proteins (H2A, H2B, H3, and H4). These histones are highly conserved and positively charged due to abundant basic amino acids, which facilitates their interaction with the negatively charged DNA. This structure resembles a "beads on a string" model [7](#page=7). 2. **30 nm fiber:** Nucleosomes are further condensed and regularly stacked, forming a fiber with a diameter of approximately 30 nm. Histone H1 plays a crucial role in this higher-order packaging by binding to DNA and facilitating the coiling of nucleosomes [7](#page=7) [9](#page=9). 3. **Condensed chromosomes:** Further compaction leads to the formation of highly condensed chromosomes, the most compact form observed during mitosis [7](#page=7). > **Example:** In vertebrates, a significant portion of genomic DNA does not code for proteins. This non-coding DNA includes introns, intragenic regions, and highly repetitive telomeric and centromeric DNA [8](#page=8). ##### 1.1.4.2 Post-translational modifications of histones Histone proteins can undergo various post-translational modifications that influence chromatin structure and gene expression without altering the underlying DNA sequence. These modifications do not occur simultaneously. Key modifications include [11](#page=11) [14](#page=14): * **Acetylation:** The addition of an acetyl group ($–COCH_3$) to specific amino acids, typically lysine, in histones. This modification neutralizes the positive charge of lysine, which can loosen chromatin structure and promote gene expression [11](#page=11). * **Methylation:** The addition of a methyl group to amino acids. * **Ubiquitinylation:** While sometimes associated with protein degradation, in the context of chromatin, it can influence chromatin compaction [11](#page=11). ##### 1.1.4.3 Epigenetic memory and X-chromosome inactivation Epigenetic memory refers to the cell's ability to retain and transmit information about gene expression patterns across cell divisions, independent of changes in the DNA sequence. Epigenetic modifications like DNA methylation, histone modifications, and chromatin remodeling are central to this process. These changes help maintain cell identity, ensuring that a keratinocyte, for instance, continues to function as a keratinocyte [12](#page=12). A prime example of epigenetic inheritance is X-chromosome inactivation in female mammals. In early embryonic development, one of the two X chromosomes in each cell is randomly inactivated to equalize gene product levels between males and females. This inactivated X chromosome becomes highly condensed heterochromatin, forming a structure known as a Barr body, and this epigenetic state is passed on to daughter cells [13](#page=13). #### 1.1.5 Nuclear pores Nuclear pores are protein-lined channels that span the nuclear envelope, regulating the transport of molecules between the nucleus and the cytoplasm. They are crucial for nucleocytoplasmic exchange [3](#page=3). ### 1.2 Nuclear organization and gene regulation The organization of chromatin within the nucleus is non-random and plays a critical role in gene regulation [15](#page=15) [17](#page=17). * **Chromosomal territories:** Each chromosome occupies a distinct region within the nucleus, referred to as its chromosomal territory [15](#page=15) [17](#page=17). * **Compartments:** Within chromosomes, chromatin clusters into transcriptionally active ("A") and inactive ("B") compartments [17](#page=17). * **Topologically associating domains (TADs):** Chromatin is further organized into large loops, often millions of base pairs long, known as TADs. These domains are generally conserved across cell types and help to insulate genes from external regulatory elements, preventing inappropriate enhancer-promoter interactions [17](#page=17). * **Sub-TAD loops:** TADs can be further compartmentalized into smaller loops, which are more cell-type specific and frequently facilitate enhancer-promoter interactions. The formation of TADs and sub-TAD loops is thought to involve the interaction of CTCF DNA-binding proteins and cohesin complexes, which bring distant chromatin regions into physical proximity [17](#page=17). > **Tip:** The structure of chromatin is not static; it is highly organized and dynamically regulated to control gene expression [17](#page=17). ### 1.3 Nucleus and mechanobiology Mechanical signals from the extracellular matrix (ECM) can directly influence the nucleus and gene expression through physical connections. Proteins like integrins, cytoskeletal elements (actin filaments, microtubules, intermediate filaments), and the LINC complex form these physical links. This transfer of mechanical signals can occur very rapidly, within milliseconds, leading to chromatin stretching and a swift upregulation of genes. Mechanical signals can also indirectly affect nuclear structure and gene expression by opening mechanosensitive ion channels in the cell membrane, nuclear pore complexes, and channels between the endoplasmic reticulum and the nuclear membrane. This modulation of ion transport and the translocation of epigenetic/transcriptional factors into the nucleus can lead to changes in chromatin structure and gene expression [18](#page=18). ### 1.4 Gene organization on human chromosomes Genes are organized on human chromosomes in a specific manner. A human chromosome consists of a short arm and a long arm. For example, the short arm of chromosome 22 is primarily composed of heterochromatin. A typical gene is composed of multiple segments called exons, which are interspersed with non-coding regions known as introns. Exons generally encode amino acid sequences that form proteins, while introns do not. During RNA processing, introns are removed through a process called RNA splicing, leaving only the protein-coding exons. The process of transcription (DNA to RNA) and translation (RNA to protein) are fundamental to gene expression [20](#page=20). --- # Nucleolus and ribosome biogenesis The nucleolus is a key nuclear organelle responsible for the synthesis and assembly of new ribosomes within eukaryotic cells [21](#page=21). ### 2.1 Structure and function of the nucleolus The nucleolus is a non-membrane-bound nuclear organelle where new ribosomes are produced. Ribosomes are composed of ribosomal RNA (rRNA) and ribosomal proteins. The nucleolus can be divided into three main functional regions [21](#page=21): * **Fibrillar center:** This is the site where the genes encoding 45S pre-rRNA are transcribed [21](#page=21). * **Dense fibrillar component:** Here, the 45S pre-rRNA is located and undergoes processing [21](#page=21). * **Granular component:** In this region, ribosomal proteins and the processed rRNAs are assembled into ribosomal subunits [21](#page=21). > **Tip:** The presence of a distinct nucleolus is a morphological characteristic often correlated with high metabolic activity in cells. Electron microscopy (EM) of such cells reveals numerous nuclear pores, a clear nucleolus, a nucleus with non-dense material (euchromatin), and abundant ribosomes and/or rough endoplasmic reticulum (RER). Light microscopy (LM) shows a pale nucleus with much euchromatin, a clear nucleolus, and granular material in the cytoplasm [24](#page=24). ### 2.2 Ribosome biogenesis in eukaryotes Eukaryotic ribosomes are composed of two subunits: a 40S subunit and a 60S subunit. The Svedberg unit (S) is a measure of sedimentation rate during ultracentrifugation [23](#page=23). * **Composition of ribosomal subunits:** * The 60S subunit contains 5S, 5.8S, and 28S rRNA molecules, along with numerous ribosomal proteins [23](#page=23). * The 40S subunit contains 18S rRNA and associated ribosomal proteins [23](#page=23). * **Synthesis and assembly:** * Ribosomal proteins are synthesized in the cytosol and then imported into the nucleus [23](#page=23). * The transcription of most rRNA molecules (excluding the 5S rRNA) occurs within the nucleolus [23](#page=23). * The genes encoding these rRNAs are located in repetitive DNA regions, often described as loops within the nucleolus [23](#page=23). * The 5S rRNA, however, is transcribed elsewhere [23](#page=23). * After their synthesis and initial processing, the ribosomal subunits are exported from the nucleus through nuclear pores. These subunits are not fully mature upon exiting the nucleus; the final assembly of the 40S and 60S subunits onto messenger RNA (mRNA) occurs in the cytoplasm [23](#page=23). > **Example:** During ultracentrifugation, a particle with a sedimentation coefficient of 40S will sediment faster than a particle with a coefficient of 20S, indicating it is larger or denser. Similarly, a 60S subunit is larger than a 40S subunit. These distinct sedimentation properties are used to classify them. --- # Nuclear envelope and nuclear pores The nuclear envelope is a double membrane system that encloses the nucleus, regulating the passage of molecules between the nucleus and cytoplasm through nuclear pores [26](#page=26). ### 3.1 The nuclear envelope structure The nuclear envelope is characterized as a double biological membrane which means it consists of two distinct lipid bilayers [26](#page=26) [27](#page=27). #### 3.1.1 Composition of biological membranes All biological membranes, including the inner and outer nuclear membranes, are fundamentally composed of a lipid bilayer. These membranes are built from amphipathic lipids, which possess both a hydrophilic (water-loving) head group and hydrophobic (water-repelling) tails. This amphipathic nature drives the formation of a bilayer structure where the hydrophobic tails are oriented inwards, away from water, and the hydrophilic heads face outwards towards the aqueous intracellular and extracellular environments [27](#page=27). * **Phospholipids** are a primary component, forming a bilayer approximately 7 nanometers thick [28](#page=28). * Other key components include glycolipids, cholesterol, and membrane proteins [28](#page=28). * The hydrophobic fatty acid tails of the lipids are held together by van der Waals bonds [28](#page=28). #### 3.1.2 Features of the nuclear envelope The nuclear envelope consists of two membranes separated by a perinuclear space [29](#page=29). * The **outer nuclear membrane** is continuous with the rough endoplasmic reticulum (RER) and is studded with ribosomes [26](#page=26) [29](#page=29). * The **inner nuclear membrane** is supported by the nuclear lamina [26](#page=26). * The **nuclear lamina** is a meshwork of proteins called lamins, which are part of the intermediate filament family. This lamina provides structural support to the inner nuclear membrane and serves as an attachment site for chromosomes [26](#page=26). ### 3.2 Nuclear pores and their function Nuclear pores are crucial structures embedded within the nuclear envelope that control and facilitate transport between the nucleus and the cytoplasm [26](#page=26) [29](#page=29). #### 3.2.1 Formation and composition of nuclear pores Nuclear pores form at sites where the inner and outer nuclear membranes fuse. Each nuclear pore is essentially a large, funnel-shaped nuclear pore complex (NPC) [29](#page=29) [30](#page=30). * The NPC is composed of numerous proteins known as nucleoporins [30](#page=30). * The number of nucleoporins can range from approximately 50 in yeast to over 100 in vertebrates [30](#page=30). * These nucleoporins are arranged with octagonal symmetry, forming a massive structure weighing about 125 megadaltons, which is roughly 30 times the size of a ribosome [30](#page=30). * Schematic models depict the transport channel within the NPC as being filled with gel-forming FG-nucleoporins [31](#page=31). #### 3.2.2 Role in transport The nuclear pores, organized within the NPC, are the primary channels for the regulated movement of molecules into and out of the nucleus. This regulated transport is vital for maintaining cellular metabolic activity. Towards the nuclear matrix, the NPC forms a basket-like structure called the nuclear basket [26](#page=26) [29](#page=29). > **Tip:** The presence of ribosomes on the outer nuclear membrane indicates its direct connection to the protein synthesis machinery of the cell. > > **Example:** Small molecules can diffuse passively through nuclear pores, while larger molecules, such as proteins and RNA, require active transport mechanisms mediated by specific signals and transport receptors interacting with the NPC. --- # Intracellular transport and nucleocytoplasmic transport This section details the general principles of intracellular transport and focuses on the active transport mechanisms through nuclear pores, including import/export signals and the role of the Ran protein. ### 4.1 General principles of intracellular transport All proteins synthesized in the cell originate in the cytosol. Their final destination is determined by transport and sorting signals, which can be linear amino acid sequences (signal peptides) or non-linear "signal patches" formed by amino acids that come together in the folded protein. Proteins lacking such signals remain in the cytosol by default. Transport mechanisms include active transport through nuclear pores, translocations, and vesicular transport [32](#page=32) [33](#page=33) [34](#page=34). ### 4.2 Active transport through nuclear pores The nuclear envelope is perforated by 3,000 to 4,000 nuclear pore complexes (NPCs). These NPCs form water-filled channels with an approximate diameter of 9 nm, allowing passive diffusion of small molecules (ions, metabolites, proteins < 20 kDa). Larger molecules, however, require energy, nuclear-specific import signals, and accessory proteins for active transport, which can temporarily enlarge the pore to about 100 nm [35](#page=35). #### 4.2.1 Visualizing intracellular transport The transport of proteins and mRNA through NPCs can be visualized using techniques like Transmission Electron Microscopy (TEM) on gold-particle-coated proteins. During nucleocytoplasmic transport, proteins generally maintain their tertiary structure. mRNA, after exiting the nucleus, unfolds in the cytosol and associates with ribosomes to initiate translation [36](#page=36) [37](#page=37). #### 4.2.2 Players in active nucleocytoplasmic transport Key components of active nucleocytoplasmic transport include: * **Receptor proteins (Karyopherins):** These receptors bind to specific signals on cargo proteins and to components of the NPC, the FG-nucleoporins. Import receptors, such as Importin-$\alpha$ and Importin-$\beta$, facilitate protein import, while export receptors, like CRM1, mediate protein export [38](#page=38) [40](#page=40). * **Ran protein:** This small GTPase is crucial for providing the energy for active transport. The Ran cycle, involving GTP-bound (active) and GDP-bound (inactive) states, is regulated by localization-dependent GEF and GAP proteins [38](#page=38) [39](#page=39) [43](#page=43). * **Ran-GEF (Guanine Nucleotide Exchange Factor):** Located in the nucleus, it promotes the exchange of GDP for GTP on Ran, activating it [38](#page=38) [39](#page=39). * **Ran-GAP (GTPase-Activating Protein):** Found in the cytoplasm, it accelerates the GTPase activity of Ran, hydrolyzing GTP to GDP and inactivating it [38](#page=38) [39](#page=39). > **Tip:** The precise localization of Ran-GAP in the cytoplasm and Ran-GEF in the nucleus is fundamental for both import and export mechanisms [43](#page=43). #### 4.2.3 Nuclear localization signals (NLS) and nuclear export signals (NES) * **Nuclear Localization Signals (NLS):** These signals typically consist of 4-8 amino acids rich in positively charged residues (Lysine and Arginine) and proline. They can be linear or bipartite and are not cleaved after import, allowing for repeated transport. Mutations in NLS sequences block nuclear import. Artificially adding an NLS to a cytosolic protein can direct it into the nucleus [40](#page=40) [41](#page=41) [42](#page=42). * **Nuclear Export Signals (NES):** These signals are typically leucine-rich sequences, often in the pattern LxxLxL, where 'x' represents any amino acid. They mediate the transport of proteins out of the nucleus. Mutating an NES can reduce the efficiency of nuclear export [41](#page=41) [42](#page=42). > **Example:** Pyruvate kinase, normally a cytosolic enzyme, can be artificially directed into the nucleus by genetically modifying it to include an NLS. Similarly, mutations in the endogenous NES of the protein p120ctn can cause it to partially remain in the nucleus instead of being exported [42](#page=42). #### 4.2.4 Ran-dependent nuclear export Ran-dependent transport is essential for exporting proteins containing NES sequences from the nucleus. The process involves [43](#page=43): 1. The protein with an NES binds to exportin and Ran-GTP [43](#page=43). 2. The complex is transported out of the nucleus [43](#page=43). 3. In the cytoplasm, Ran-GTP is hydrolyzed to Ran-GDP, promoting the dissociation of the cargo protein from exportin [43](#page=43). 4. Exportin returns to the nucleus [43](#page=43). --- ## Common mistakes to avoid - Review all topics thoroughly before exams - Pay attention to formulas and key definitions - Practice with examples provided in each section - Don't memorize without understanding the underlying concepts

| Term | Definition | |------|------------| | Nucleus | The central organelle of a eukaryotic cell, enclosed by a double membrane, containing the genetic material (DNA) organized as chromatin or chromosomes. | | Nuclear envelope | A double membrane surrounding the nucleus of a eukaryotic cell, perforated by nuclear pores that regulate the passage of molecules between the nucleus and the cytoplasm. | | Nuclear pore | A protein-lined channel in the nuclear envelope that regulates the transport of molecules between the nucleus and the cytoplasm. | | Nucleolus | A dense structure within the nucleus where ribosomal RNA (rRNA) is transcribed and ribosomal subunits are assembled. | | Chromatin | The complex of DNA and proteins that forms chromosomes within the nucleus of eukaryotic cells; it exists in two forms: euchromatin and heterochromatin. | | Euchromatin | The less condensed form of chromatin, typically found in the nucleus, which is transcriptionally active and accessible to the cellular machinery. | | Heterochromatin | The more condensed form of chromatin, typically found in the nucleus, which is transcriptionally inactive and tightly packed. | | Karyoplasm | The substance that fills the nucleus, analogous to the cytoplasm that fills the cell; it contains chromatin, the nucleolus, and various enzymes and molecules necessary for nuclear functions. | | Histones | Proteins around which DNA is coiled to form nucleosomes, a fundamental structural unit of chromatin. They play a crucial role in DNA packaging and gene regulation. | | Nucleosome | The basic structural unit of chromatin, consisting of a segment of DNA wound around eight histone proteins. | | Post-translational modification | The chemical modification of a protein after its translation from mRNA, which can affect its folding, stability, localization, and function. Examples include acetylation and methylation of histones. | | Epigenetic memory | The ability of cells to retain and transmit information about gene expression patterns across cell divisions without altering the underlying DNA sequence, mediated by epigenetic modifications. | | Barr body | An inactivated X chromosome in female mammals, which is condensed into heterochromatin and serves as an example of epigenetic inheritance. | | Chromosomal territories | Discrete regions within the nucleus occupied by individual chromosomes, maintaining a non-random spatial organization. | | Mechanobiology | A field of study that investigates the physical forces acting on cells and tissues and how cells respond to these mechanical stimuli, including their influence on nuclear structure and gene expression. | | Integrins | Cell surface receptors that play a role in cell adhesion and mechanotransduction, linking the extracellular matrix to the cell's internal cytoskeleton and signaling pathways. | | LINC complex | (Linker of Nucleoskeleton and Cytoskeleton) A network of proteins that connects the nucleus to the cytoplasm, mediating mechanical signaling from the exterior to the interior of the cell. | | Transcription | The process of synthesizing RNA from a DNA template, a key step in gene expression. | | Translation | The process of synthesizing a protein from an mRNA template, where the genetic code is decoded into an amino acid sequence. | | Exons | Coding sequences within a gene that are transcribed into mRNA and are eventually translated into protein. | | Introns | Non-coding sequences within a gene that are transcribed into pre-mRNA but are removed during RNA splicing before translation. | | RNA splicing | The process of removing introns from pre-mRNA and joining exons together to form mature mRNA, which is then translated into protein. | | rRNA | Ribosomal RNA, a component of ribosomes responsible for protein synthesis. | | Ribosomal subunits | The two components (large and small) that make up a ribosome, which assemble on mRNA to perform translation. | | Nuclear Pore Complex (NPC) | A large protein complex that spans the nuclear envelope and regulates the transport of molecules between the nucleus and the cytoplasm. | | Nucleoporins | Proteins that form the nuclear pore complex. | | Intracellular transport | The movement of molecules and organelles within a cell. | | Post-translational transport | Transport of a protein to its cellular location after its synthesis on free ribosomes in the cytoplasm. | | Co-translational transport | Transport of a protein to its cellular location that occurs simultaneously with its synthesis on ribosomes attached to the endoplasmic reticulum. | | Signal sequence | A specific sequence of amino acids within a protein that directs it to a particular cellular compartment or destination. | | Signal peptide | A type of signal sequence, typically found at the N-terminus of a protein, that targets it for translocation across a membrane or to a specific organelle. | | Signal patch | A non-linear signal sequence formed by amino acids that are brought together in the three-dimensional structure of a folded protein, directing its transport or localization. | | Nuclear Localization Signal (NLS) | An amino acid sequence that targets a protein for import into the nucleus. | | Nuclear Export Signal (NES) | An amino acid sequence that targets a protein for export from the nucleus. | | Karyopherin | A class of proteins that act as transport receptors for molecules moving between the nucleus and the cytoplasm. Importins facilitate import, and exportins facilitate export. | | Ran | A small GTPase protein that plays a critical role in regulating nucleocytoplasmic transport by controlling the directionality of import and export. | | Ran-GAP (GTPase Activating Protein) | A protein found in the cytoplasm that stimulates the GTPase activity of Ran, promoting the hydrolysis of Ran-bound GTP to GDP. | | Ran-GEF (Guanine Nucleotide Exchange Factor) | A protein found in the nucleus that promotes the exchange of GDP for GTP on Ran, generating the active, GTP-bound form of Ran. |