TOPIC 6: Nuclear Structure PDF

BLOCK III TOPIC 6: NUCLEAR STRUCTURE INDEX: 4. Nucleoplasm 5. Nucleoskeleton 5.1. General features 5.2. Components of the nucleoskeleton 5.3. Functions 6. Nuclear envelope 6.1. Components 6.2. Net of Networks 6.3. LINC complex INTRODUCTION I...

BLOCK III TOPIC 6: NUCLEAR STRUCTURE INDEX: 4. Nucleoplasm 5. Nucleoskeleton 5.1. General features 5.2. Components of the nucleoskeleton 5.3. Functions 6. Nuclear envelope 6.1. Components 6.2. Net of Networks 6.3. LINC complex INTRODUCTION In eukaryotic cells, the nucleus is the organelle responsible for maintaining the integrity of DNA and for controlling cellular activities such as metabolism, growth, and reproduction by regulating gene expression. It is a double-membraned organelle containing nuclear structures, such as chromatin, nuclear bodies, and nuclear matrix. 4. NUCLEOPLASM/KARYOPLASM/NUCLEAR JUICE The nucleoplasm is the inner media of the nucleus (like the cytoplasm of the nucleus), a gel-like matrix in which components of the nucleus are located. It allows chemical reactions to be carried out in the nucleus, being the matrix in which chemical reactions happen. It is composed of various substances and molecules including nucleic acids (DNA, RNA…), proteins necessary for nucleic acid metabolism, products derived from glycolysis (ATP, NAD and Acetil Co-A) and ions (K+, Na+ and, overall, Ca++ and Mg++). 5. NUCLEOSKELETON 5.1 GENERAL FEATURES The idea of a nucleoskeleton emerged in the 1970s, making it quite a new concept which initially was a bit controversial. Many thought its only function was to provide mechanical support and functional organization to the nucleus. The nucleoskeleton was discovered as the residual nuclear protein fraction left after the removal of chromatin with high salt solutions. Back then its main function was believed to be the organization of the genome in the nuclear volume and in some way the regulation of gene expressions. Nowadays we define the nucleoskeleton as the residual content left after the elimination of the nuclear envelope (NE), chromatin and other residual components. This elimination happens by sequential treatment with non ionic detergents, nucleases and buffer solutions. The main component left is a kind of filament, to be precise, intermediate filaments. These filaments are extremely strong and stable making them difficult to destroy. These intermediate filaments in a way have a continuation with the cytoskeleton and some other proteins are also attached or associated with it. It is the major site for the chromatin function ( transcription, splicing, DNA repair…) and it provides mechanical support and functional organization: Organizes the genome in nuclear volume Participates in gene expression regulation Retain constituents of nuclear regions and domains in place 3D organization of the general nuclear structure It is a fibrogranular network of non histonic proteins 5. 1. 1. TYPES OF PROTEINS IN THE NUCLEOSKELETON ○ Lamins: A and B, their lack closely associated with diseases. ○ Lamin binding proteins. ○ Actin: a bit different from the one in the cytoskeleton, it is also a microfilament but it is more globular ○ NuMa (Nuclear Mitotic apparatus protein): essential in the shape of the nucleus and also in gene expression regulation. ○ Myosin: motor protein of the cytoskeleton, but is also in the nucleus. ○ Emerin: structural role, important in a muscular disease. ○ Titin. ○ Residual content of nuclear envelope 5. 2. COMPONENTS OF THE NUCLEOSKELETON The nucleoskeleton is the cytoskeleton of the nucleus. Nucleoskeleton is a three-dimensional fibro-granular net(work) of non-histone proteins associated with some globular proteins (actin filaments), which extends from the inner membrane of the nuclear envelope almost all the way into the nucleoplasm (it is distributed through the whole nucleus). It associates with globular proteins related to chromatin, RNA and its processing, this is, the nucleoskeleton is key in the expression of genes. It is very dynamic, so it can reassemble and change rapidly. The nucleus has intermediate filaments inside, which are forming part of the nucleoskeleton. There are four defined areas or domains in the nucleoskeleton: Nuclear lamina or inner fibrous lamina: It’s the peripheral nucleoskeleton. Some regions are really closely associated with LAD (lamina associated domains) and with the nuclear envelope scaffold. Nuclear matrix/karyoplasm/nucleoplasm, and associated domains: It’s the inner nucleoskeleton, the inner network of globular proteins. They can be anchored to specific chromatin regions called S/MAR (Scaffold Matrix Associated Regions). Pore linked filaments. NAD-Scaffold (Nucleolar Associated Domain Scaffold); Areas in the nucleoskeleton that are used to keep the chromatin associated with the nucleolus in place. While the cell contains a cytoskeleton that provides structural support and a means of transport throughout the cytoplasm, the nucleus also contains (nucleolar) fibrous scaffolding (andamio) surrounding the nucleolus. This nuclear scaffold is going to keep together all the machinery needed by the nucleolus to make the pre-ribosomes. Both nucleoskeleton and cytoskeleton are both networks which together create one big net. That’s why the whole net of the cell is called “net of networks”, because it is one net made up by two smaller networks (nucleoskeleton and cytoskeleton) that are associated with each other. Nucleoskeleton and cytoskeleton form networks linked by complexes at the nuclear envelope, so they are attached to each other. Plasma membrane contains adhesion complexes. These adhesion complexes interact with neighbouring cells or ECM (extracellular matrix, a network consisting of extracellular macromolecules and minerals that provide structural and biochemical support to surrounding cells). Their interactions send signals and transmit mechanical forces to the cytoskeleton. As a result, cytoskeleton attach to NE (nuclear envelope)-LINC (linkers of the Nucleoskeleton to the Cytoskeleton) complexes, through nesprin, SUN domain containing proteins and KASH-containing domains. Then LINC complexes transmit signals to chromatin and nucleoskeleton. That is what we call mechanotransduction 5. 2. 1. NUCLEAR LAMINA The nuclear lamina is a dense fibrillar network of intermediate filaments. It makes extensive contacts with integral proteins of the inner nuclear membrane (extremely associated to INM of the nuclear envelope) and with chromatin. It takes part or participates in nuclear positioning, in mechanotransduction, in nuclear stability, in the 3D organization of the chromatin and in the regulation of chromatin binding proteins. It is present in all metazoans (animals), but the composition differs between tissues. It consists in various nuclear lamins and associated proteins. COMPOSITION ○ It is formed by Lamins, specially lamins A and B (lamins A are all over the nucleus but Lamins B more specifically in the nuclear lamina). ○ Lamins bind to the inner membrane of the nuclear envelope with Lamin binding proteins (nuclear receptors) NUCLEAR LAMINS Type V intermediate filaments are present only in metazoans(animals). Most cells express A and B type lamins, although there is also a C type. LMNA gene codes for lamins A and C, and these create stiffness, making the nucleus susceptible to damage on mechanical stress. LMNB gen codes for lamin B, which is essential for cell viability. Any mutation in any of those genes, would provoke a disease. These nuclear lamins are bound to NE through interaction with specific receptors that are synthesized in the ER. Thanks to vesicular trafficking , proteins are being synthesized in the ER membrane and the little by little are going to get to the correct place due to membrane diffusion. They are classified according to their association with the INM during mitosis → INM bound and INM not-bound. LAMIN BINDING PROTEINS AND ASSOCIATED PROTEINS LOCATION It spreads under the inner nuclear membrane, associated to LAD and nuclear envelope scaffold. It spreads among NPC (nuclear pore complexes), and it is interrupted in the places in which there are NPC. It is closely related to the INM (inner nuclear membrane) of the nuclear envelope. Remember! : Nuclear lamina is extremely important for the 3D organization of the chromatin, is going to allow the chromatine to condensate and get a 3D organization within the nucleus. DISEASES RELATED TO THE NUCLEAR LAMINA When defects occur in lamins (mostly lamin A) and/or lamin binding receptors, these cause laminopathies (a wide spectrum of diseases associated with mutation in lamin genes). When people lose nucleoskeleton DNA binding, diseases arrive. This happens because the defects occurring in lamins and/or lamin receptors severely affect the morphology of the nuclear envelope and the 3D organization of DNA, and therefore, genes and gene expression. Among these laminopathies we can find: - Skeletal muscle atrophies (Emery-Dreyfuss muscular dystrophy) - Fat tissue atrophies (Familial Partial lipodystrophy) - Nervous system atrophies (Peripheral Nerve System defects) - Progeria: it is the most known laminopathy. Progeria means premature aging. It is a rare continental disorder caused by mutations in a lamin. They have rare nuclear morphology, because the nucleoskeleton is defective. A normal nucleus and a nucleus with changes in lamin A, related to changes in the 3D organization of chromatin and the gene expression (replication, transcription…) 5. 2. 2. NUCLEAR MATRIX In an interphase nucleus, chromatin is present in a relatively condensed state. Thanks to the nuclear matrix the chromosomes in the nucleus are organized and each one has its own location. When the histone proteins and the lipids are removed from the nucleus, the remaining fibrous mesh of proteins is called the nuclear matrix. Nuclear matrix gives the structural and architectural stability to the nucleus. It is associated or anchored to S/MAR, DNA/chromatin sequences or domains. COMPOSITION Nucleolar scaffolds: these are supportive elements that are parts of the nuclear matrix, and they give structural support for the rest matrix (“andamio”). S/MAR (Scaffold or Matrix associated domains/regions): are DNA sequences associated to the matrix or to the Scaffolds, sometimes directly, others with proteins. STRUCTURAL ORGANIZATION OF THE GENOME: Chromatin domains associated to the nucleoskeleton Chromatin domains are the spaces in which entire chromosomes are organized inside the nucleus. This way each chromosome is packed without intermingling with others and thus keeping an order inside the nucleus, which permits the proper function of the cell regarding the synthesis of proteins and mitosis among other processes. Among the types of domains there exist the following: TAD (Topologically Associated Domains): (Large chromatin domains that interact more within themselves than with adjacent domains) ○ They are formed by groups of chromatin loops and thanks to them is ensured each cell type specific gene expression. ○ They separate the promoters of one TAD from the enhances of different TADs, and on the other hand, the interactions of promoters and enhances of the same TAD are promoted. That means that those domains only have interacctions within their own territory. ○ The limits of the regions, the boundary regions are very enrich in CTCF and cohesin ○ Areas of chromosomes that interact with themselves more than with any other chromosome in the nucleus. Basically the space each different chromosome takes. This TADs get in a higher level of compactation forming chromatin compartments: A compartment: open chromatin state (loose) ○ High content of active genes ○ Early transcript genes (quick answers) ○ Permissive transcriptional enviroment  ○ Located preferentially in the central region and close to NPCs B compartment: closed chromatin state (compacted) ○ High contact of silenced genes (non active genes) ○ Late replication timing ○ Located preferentially at the periphery interacting with lamin LADS There are some specific regions in the TADs that they are going to anchor to the nuclear matrix; they are rich in CTFT protein (a type of binding protein) which is going to help binding the loops S/MARS (Scaffold/matrix attachment regions): The area in which TAD borders overlap and chromatin binds to the nuclear matrix. These are associated with the scaffold specifically in mitosis. Each chromosome has its own place and they keep those places after the cell division. Nowadays we still do not know how chromosomes know where to go, but we know that the cytoskeleton plays a role in this process. LAD (Inner lamina associated domains): Areas in which chromatin condenses and associates to the internal nuclear membrane. The is a control of passing from a chromatin to a heterochromatin depending if those areas of chromatin must be transcript or not. There is heterochromatin surrounding the nucleolus and stabilized by NADs. There are two types according to the type of chromatin associated: ○ cLAD(constitutive): These areas are in direct contact to the INM ○ fLAD(facultative): These areas are the continuations of consecutive LAD but are not in direct contact to the INM. These are domains of the chromatin associated with the lamin that a certain point are going to be transcribed. NAD (Nucleolar associated domains): Areas associated with the perinucleolar matrix (the outside zone of the nucleolus). LADs and NADs are associated and are poor in genes. CHANNEL MODEL Why are these channels important? Imagine, we have here the DNA genes that must be transcribed to RNA. But this RNA, to be transcribed, needs polymerases. And we will finally get our messenger RNA. But this messenger RNA must go out the nucleus to reach the cytoplasm. Imagine, if this machinery, these RNAs would be in the center of the nucleus, it would be very slow and not very efficient actually. The transfer of those RNAs to the cytoplasm. But we have a scaffold forming a kind of highway. A highway, the messenger RNA to exit the nucleus. And in fact, to the scaffold forming those channels, connecting directly to the nuclear pore complex. 5. 3. FUNCTIONS OF THE NUCLEOSKELETON We have the constitutive ones (permanent) and the facultative ones (only when the gene must be transcripted). The nuclear lamina is specially important because it keeps the shape of the nucleus. If the cell has a disease, lamines are mutated and they cannot polymerize correctly, so the hall nucleus changes, in some cases affecting the gene expression.. According to the structural level: - Nuclear shape and size: the nucleoskeleton provides structural stability to the nucleus, also incredible flexibility and shape. The change of the nucleus shape is associated with diseases. It keeps the nuclear shape and size. - Chromatin and other structural elements organization: Formation of loops and gene expression is thanks to the interaction of S/MAR regions and Nuclear-Scaffolds; they reorganize the DNA depending on the needs of the cell. The S/MARs change the form of the matrix for transcription and for cytoplasmic transport. For example, the nuclear matrix reorganizes to allow the RNAm transport from the nucleus to the cytoplasm. - Chromatin folding and loop modeling: (S/MAR, TAD, LAD and NAD). Abling the model of organization of the chromatin (the different domains categorized above).  In this part is essential the participation of the nuclear matrix. Chromatin and other structural elements organization: Functional functions of the nucleoskeleton (related with replication and gene expression) - Mechanotransduction: the proteins in the nuclear envelope are connected with the nucleoskeleton that is at the same time linked to the cytoskeleton. When the cytoskeleton receives a signal from the outside it passes the signal to the nucleoskeleton through transmembrane proteins in the nuclear envelope. This function is very important, as these signals coming from the outside can be related with gene expression, so when a protein needs to be expressed and the signal arrives to the nucleus by the mechanotransduction. It can cause the location of the nucleus to change. - Facilitating the machinery of gene transcription, replication and regulation by maintaining the chromatin organized in loops (which create spaces by which those processes can be fulfilled). By maintaining the loops, it is going to favor the access of those genes to the machinery and to all the molecules the gene needs to transcribe. Then if the cell receives a signal to express certain genes, the smart proteins and the cromatine will move the heterochromatin so it becomes euchromatin. That way, all the machinery will be able to reach that chromatin. - The nucleoskeleton´s inner matrix is an anchoring site in which the molecular complexes needed for mechanisms regarding chromatin can be stored until activation. Also, the function of activation of transcription and anchoring of S/MAR to the nuclear matrix. - In response to the external stimuli, S/MAR proteins can modulate the chromatin orchestration of genes causing the activation of one gene, while repressing others. Anchoring function for the S/MAR (the area that is binded to the nucleus) and activation of transcription ( transcription starts from these complexes) Anchoring a chromatin loop which  serves as a way of insulating (in said loop) a gene that needs to be either repressed or stimulated. The formation of transcription machinery in the anchored site of the S/MAR. They are also, anchoring sites for replication complexes, viral replication, transcription, etc. - Structural transcription factors (CFTF and COHESIN) cooperate the partition the genome in looped domains - Anchor site of the looped domains, include, exclude or even lok the target gene's topological domain. In the picture we can see the nuclear matrix, the chromatin and two of these facultative S/MARS that are anchoring the chromatin to the nucleoskeleton (They are stabilizing the loop). Then we have the gene, and close to it, we have the facultative S/MAR. When this gene has to be transcribed the S/MAR is going to anchor the genes to all the machinery that is aggregated in that region of the nucleoskeleton. So this gene will be so close that the transcription will be very effective. Once the gene has been transcribed, the S/MAR will separate from the nucleoskeleton and the loop will recover the previous shape In this function of the nucleoskeleton two factors are also involved: the CTCF, which is just a condensin (says where the loop stops beeing formed), and we also have the cohesin that helps to make the loops and to get the facultative. A PROPOSED MODEL FOR THE SELECTIVE USE OF S7MAS FOR THE TRANSCRIPTION/REPLICATION REGULATION: 6. NUCLEAR ENVELOPE It is the envelope surrounding the nucleus, so it is the equivalent of the plasma membrane of the cell. FUNCTIONS - Shields the genome from cytoplasmic components not allowing molecules to enter the nucleus - Anchorage for chromatin and the cytoskeleton - Chromatin organization, different chromosome territories - Gene regulation, for example the calcium sensitive gene expression regulation - Nucleus-Cytoplasm transport of substances - Mechanotransduction - Transcription and RNA maturation separation from translation 6.1. COMPONENTS OF THE NUCLEAR ENVELOPE: Nuclear Membrane: the nuclear membrane has two phospholipid bilayers the inner and the outer nuclear membranes. Those membranes are quite impermeable to ions and small molecules. All the components inside the nucleus have to be controlled and they cannot allow molecules going inside the nucleus because it would be a disaster. When seeing a microscopy image of these we can identify them by looking at the nuclear components. On one side we can see parallel filaments while on the other side we can find a basket shape. The first one belongs to the cytoplasm while the latter belongs to the nucleoplasm letting us know where the outer and inner nuclear membrane will be. Both membranes are very different from one another, with different sets of transmembrane proteins. Moreover, they don’t communicate with each other because the perinuclear space is between one and the other. The nuclear pore opens a space from the cytoplasm to the nucleoplasm that interrupts the perinuclear space. The outer membrane is continuous with the endoplasmic reticulum so the cisterns for both are connected, but they aren enriched in different proteins (Nucleoporins, Specific INM proteins -NET- and specific ONM proteins). Some proteins that we can find in the membranes of the nuclear envelope have something in common but many of them cannot be found in the nuclear reticulum. They are synthesized here but they fluid because of the fluidity of the membranes to reach them both and go to the ones who are adequate to those proteins. Mutations or issues with the quantity of proteins from the nuclear envelope extremely influence human physiology causing many diseases, a lot of which are associated with neurodegenerative diseases. The nuclear envelope breakdowns and reassembles in mitosis. The EN associated proteins in prophase to translocate to chromosomes and to distribute with fragmented ER and in anaphase, to around condensed chromatin and the nuclear lamin t periphery. The lumenal space separates the ONM and the INM and s continous with the ER (endoplasmic retiulum) Outer nuclear membrane (ONM) Ribosomes are attached to this membrane. We have many proteins in the ONM but the most important ones are NESPRINS, these proteins possess a domain called KASH. This domain is so important because it will be the one to connect to the transmembrane proteins in the INM, this way signals transmitted through the cytoskeleton from the cytoplasm will be able to pass to the nucleus. NESPRINS are connected to the cytoplasmic side by actin (Calponin homology domain interacts with actin), dynein, microtubules (Spectrin repeats interacts with microtubules), kinesin and intermediate filaments. They also connect the nuclear envelope with both the cytoskeleton and the nucleoskeleton to transfer proteins,, they can do this because they have the ability to bind to a protein called SUN that is located in the perinuclear space, in a way providing an adhesion point between the cell exterior, through the cytoskeleton, with the interior of the nucleus and all the transcription machinery. LINC COMPLEX Inner nuclear membrane (INM) It faces the nucleoplasm and is connected to the ONM via pore membrane. The integral protein access are controlled by NPC and the integral INM proteins interact with chromatin or nuclear lamina, reguate nuclear envelope spacing, proteins with SUN domain interact with ONM/KASH and other integral membrane proteins (MAN1, LAP2, LBR). Nesprin interacts with the INM thanks to the KASH domain which also interacts with the SUN domain transmitting the signal from the cytoskeleton to the INM and from there to the nuclear lamina. This nuclear lamina will transmit the signal directly or indirectly to the chromatin which can change the 3D organization of the chromosome. This change also affects gene expression. Nuclear Pore Complex (NPC) Regulates the export and import between the nucleus and the cytoplasm Perinuclear space or perinuclear cistern: It’s the space between the inner and outer nuclear membrane (30-50 nm) and it will function as a storehouse for calcium, which will be essential for many reactions that take place within the nucleus such as DNA replication and transcription or nuclear signaling. Also it influences the NPC size and permeability and transmit osmotic forces tothe nucleus (because of the continous exchange of ions) Is continous with ER lumen. To get the calcium ions into and out of the perinuclear space the nuclear envelope will use pumps and channels (Na/Ca exchanges helps calcium regulation). Due to the fact that there is a large amount of calcium in the nuclear envelope new ions coming from the cytoplasm will be going up the gradient meaning they will need pumps. Meanwhile, as calcium is needed in the nucleoplasm, channels will be used for the exit of the ions since the concentration is lower than in the perinuclear space. Nonetheless, calcium can also pass through the nuclear pore complex by passive transport. Overview of the nuclear envelope 6.2. NET OF NETWORKS: Thanks to the interactions between the SUN and KASH domains a net of network is able to form between the nucleoskeleton and the cytoskeleton. Signals from the cytoplasm can be sent to the chromatin directly because in the chromatin we have regions called S/MARs which are related to scaffolds. Without S/MARs many of the signals would be lost. This network will receive signals and will also be able to sense mechanical stresses or changes in the shape of the cell. Cells are sensitive to matrix rigidity and geometry. This allows the nucleus to react, for example starting the process to create new proteins if needed. The SUN and KASH domains together create the LINC complex, this complex is the link between the cytoplasm and the nucleoplasm. Mechanical states modulate cytoskeleton-nucleus link, they modulate 3D organization of chromosomes to regulate gene expression. 6.3. LINC COMPLEXES Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) consist in macromolecular assemblies that span the NE and physically connect the nuclear lamina to different components of the peripheral cytoskeleton and they structurally support the nucleus. They have a MECHANOSENSORY ROLE. Sun proteins form heterotrimeric complexes that interact with KASH domains. LINC complexes are formed by the direct interaction, within the perinuclear space, between SUN proteins and Nesprins,. The connections are mediated by INM and ONM proteins.