Neuron Cytoskeleton PDF

Translated from Catalan to English - www.onlinedoctranslator.com T3. NEURONAL CYTOSKELETON 1. REMINDER Today we focus on explaining the particularities of the cytoskeleton of neurons, commenting on specificities of the neuron, i...

Translated from Catalan to English - www.onlinedoctranslator.com T3. NEURONAL CYTOSKELETON 1. REMINDER Today we focus on explaining the particularities of the cytoskeleton of neurons, commenting on specificities of the neuron, intracellular transport and polarity. It will be a reminder of concepts we may already have and some new ones. In a neuron will we find the same cytoskeleton composition as in the rest of eukaryotic cells? - Answer:Yes, we will work with the same types of cytoskeleton (intermediate filaments or neurofilaments, tubulin microtubules and actin microfilaments). What type of cytoskeleton has a basically structural function? - Answer:Neurofilaments are basically structural, they do not participate much in intracellular mobility. What type of cytoskeleton is used for transport over short distances? - Answer:Actin microfilaments participate in the mobility of intracellular organelles at short distances. On the other hand, microtubules participate in long-distance mobility. 2. INTRODUCTION 2.1. STRUCTURE OF NEURONS Some of the neurons we can find in our brain have a high complexity in terms of morphology. In addition, the different neurons are communicating and transporting components through the different compartments. So there has to be something that makes this structural complexity possible and maintained, and that's what thecytoskeleton. With regard to the structure of the neuron, it was already seen from the time of Ramon and Cajal thatthe neurons exhibited a great diversity of morphologies, which were maintained over time and conserved between species. Furthermore, distinct populations of neurons defined by morphology could be differentiatedusing different markers biochemical and immunological. In addition, themorphology of neurons was correlated with functional properties, the structures of the morphology of the neurons was well preserved, it was constant and maintained. The finer analysis of the structures of the cell demonstrated the existence of very small structures that participate in the connectivity between neurons (synapses). These structures were the dendrites. The growth cone was also described. This diversity in the structures of neurons can be reproducedin vitroplanting neurons at different stages of development that in a culture can differentiate into very diversified structures. we can see a diagram of the different stages through which differentiation takes placein vitro of neurons.At first we observe a dendrite, but as time passes the different structures appear. 2.2. NEURONAL CYTOSKELETON The neuronal cytoskeleton represents a25% of the total proteinof the neuronal cell, a very high percentage that makes us see its importance. Cytoskeletal proteins are determinants for adetermine the morphology of the neuron , since they allow the organization of the interior of the cell, compartmentalize it and guarantee its functions. This is very important since the neuron is onepolarized cell, that is, it has two sides (axon and somatodendritic component) with completely different functions. We must also emphasize the importance of the cytoskeleton in the intracellular transport, since the distance that a neuron can cover can be extremely large. There is a great distance between the neuronal compartments that requires a complex transport system of macromolecules and organelles. As we already know, there are 3 main components of the cytoskeleton of any cell (neurofilaments or intermediate filaments, actin microfilaments and tubulin microtubules) that differ by chemical, physical characteristics, composition, thickness... Thus, in the diagram below, we see that the intermediate filaments have a greater capacity for deformation and resistance to rupture, which indicates their fundamentally structural function. Commenting on the dimensions of the 3 types of cytoskeleton, we can say: - Theneurofilaments (NFs)they are several microns long and about 10 nanometers thick - Themicrotubulesthey are longer and thicker; they can measure up to 100 microns and be about 25 nanometers thick. - Themicrofilamentsof actin are the finest; they measure several microns and have a thickness of about 9 nanometers. 3. INTERMEDIATE FILAMENTS – NEUROFILAMENTS 3.1. WHAT ARE THEY? Theneurofilamentsare one of the types of intermediate filaments that can exist. Their structure has the shape of a rope, and they are characterized by being a highly polymerized and very stable structure. The intermediate filaments fundamentally confer mechanical resistance, and participate in the structural support of the nuclear membrane, in the integrity of the cells within the tissues and in the formation of structural and functional barriers of the skin, hair and nails. It must be said that they do not have any type of engine, since they have no function of transport 3.2. STRUCTURE OF INTERMEDIATE FILAMENTS The structure of the intermediate filaments begins with the monomers, which are the basic structure. 2 monomers combine to createdimersin the form of a double helix. 2 dimers form onetetramer,and two tetramers form one protofilament. 8 protofilaments joined in parallel form one filament, which has the rope structure we talked about, which is very resistant. It must be said that neurofilaments they are not polar, since when the two dimers form the tetramer they join the head of one with the tail of the other, forming a symmetrical structure. 3.3. DISTRIBUTION OF INTERMEDIATE FILAMENTS IN THE ORGANISM Human intermediate filament proteins (IFs) are encoded by 70 genes. We can classify IFs into 6 types based on sequence similarities. In the case of neurons we will have the following proteins:NF-L(NF light),NF-M( NF medium),NF-H(NF heavy), alpha-internexiniperipheryHowever, apart from the NFs we will have other proteins such as:vimentin( neuronal and glial precursors); GFAP astrocytes and some Schwann cells;loss(Smooth muscle cells in the vasculature);nuclear laminins( nuclear envelope);nestin(neuroectodermal precursors in brain development). IFs are tissue-specific filamentous structures. In the following image we can see how the intermediate filaments are distributed throughout the body. 3.4. SPECIFIC STRUCTURE OF NEUROFILAMENTS Referring to the structure of neurofilaments in particular, we must comment that we have 3 main components, all of them homologous with a certain diversity in sequence. These are theshort chain neurofilament (NEFL), medium chain (NEFM) and heavy chain (NEFH). The latter has oneKSP domainwith many potential phosphorylation sites. We can also find incorporated other homologous molecules such as internexins or peripherins. The area with the most homology between the different types of filaments is a domain of approximately 300 amino acids calledRod domain. So neurofilaments are fundamentallyheteropolymersof NFL with either NFM or NFH units (not both). In addition, the tails of all 3 forms are found protruding through the external region of what would be the structure of the filament itself (the one with 8 protofilaments). These tails allow cross interactions between neurofilaments, but also with other structures. Specifically: - NFM and NFH of the different NFs can create cross-bridges, especially important for axonal projections. - NFM help in longitudinal extension - NFH interact with microtubules and other components of the cytoskeleton. 3.5. NEUROFILAMENTS IN DISEASE It is also important to mention that due to the ability to give a flexible and at the same time firm structure to the cell, neurofilaments are particularly important in axons that have to connect with distant regions, usually myelinated axons. In the following image we see an electromicrograph of a section of an axon in which we see a series of small dots which are the neurofilaments, and some larger dots which are the microtubules. We see that there is a great abundance of neurofilaments compared to microtubules, indicating their importance in maintaining the structure of the axon. Therefore, alterations in the structural composition of neurofilaments can lead to incorrect functioning of neurons. When KO models have been made of the different chains of the neurofilaments, they show deficits in the stability of the axons. In addition, mutations in neurofilaments are linked to diseases such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and Alzheimer's and Parkinson's diseases. In addition, it is hypothesized that the presence of neurofilaments in the blood or in the cerebrospinal fluid can be potential biomarkers of a large number of neurodegenerative diseases. It is thought that these neurofilaments may appear in blood or CSF before symptoms appear. 4. MICROTUBULES 4.1. GENERALITIES The next type of cytoskeletal element we will discuss are themicrotubules Microtubules are an extensive network of filaments that extend throughout the entire neuron. In addition, they have a very important role in the development and maintenance of theneural extensions. They also provide onestructural and transport networkfor organelles and associated macromolecules. They are made up of filaments made up of dimers of α or β tubulin with a polar structure. The molecular motors that use this structure as a transport route between the different molecular compartments are thekinesin and dynein motors. In addition, they also existmicrotubule associated proteins (MAPs),which are responsible for stabilizing microtubules and creating connections between adjacent microtubules. 4.2. STRUCTURE OF MICROTUBULES As we have already advanced before, microtubules are formed by polymerizations of a and β tubulin, two molecules of about 55kDa and which have 40% sequence identity between them. On the one hand, α-tubulin binds GTP inside it. On the other hand, β-tubulin binds either GTP or GDP, and is also able to hydrolyze it. The filamentous structure of microtubules is formed from the sequential union of α and β tubulin forming what is known as aprotofilamentGroups of 13 protofilaments form the cylindrical structure we call a microtubule. The fact that the protofilaments are always joined with the same non-symmetrical orientation means that one of the ends of the microtubule contains only α-tubulin molecules and the other end has only β-tubulin. Here we do find a structure polar, which is important for its function. 4.3. NUCLEATION OF MICROTUBULES In order to form the microtubule through α and β tubulin, a can be producedspontaneous nucleationdepending on the they form the microtubules by themselves. For another gulled and facilitated by other structures. 4.4. ELONGATION OF MICROTUBULES Due to the fact that microtubules are non-symmetric and polar structures, a phenomenon occurs in which there is one end in which growth due to the formation of new dimers of α and β tubulin is much faster than in the other. So, we find: oneextreme +in which the β-tubulin is exposed in which the formation of dimers is facilitated, and which consequently exhibits growth. it happens thanks to the fact that this end is able to hydrolyze the GTP it incorporates. GTP is preferentially incorporated in the positive side, and stabilizes the polymer. oneextreme –in which α-tubulin is exposed. In this extr incorporation of dimers and GTP is not facilitated, so that instead of showing growth, it shows a loss, a decrease due to more GDP. This phenomenon means that there is a balance between growth and decline that ends up defining themicrotubule mobility towards the positive side. 4.5. DYNAMIC INSTABILITY In the case of microtubules, due to the requirements of the neuron's own physiology there is onedynamic instabilitywhich means that due to the action of some proteins in concert moments it may happen that there is one catastrophe,that is to say a break of the positive end region due to a sudden loss of tubulin dimers. After the catastrophe, the length of the microtubule is greatly shortened. It must be said that this is a regulated process that allows the size of the microtubule to decrease quickly and effectively. 4.6. POST-TRANSLATIONAL MODIFICATIONS OF TUBULIN There are post-translational modifications (PTMs) of tubulin that contribute to the functional diversity of microtubules. These modifications affect athe dynamics, organization and interactions of microtubules with other cellular components. Furthermore, several modifications are localized to specific microtubule domains, thus demonstrating a largeheterogeneity. Finally, it must be said that these modifications vary according to time, cell type, subcellular compartment and physiological state. These post-translational modifications can be denitrosinations, acetylations, phosphorylations, polyglutamations , etc. 4.7. ORGANIZATION OF MICROTUBLES IN THE NEURON Throughout the life of the neuron, and unlike what happens in other cell types, there is not always a clear and continuous point of organization of the set of microtubules. Thus, in early stages of development in a modelin vitrowe can see that initially we find a microtubule organizing center (MTOC) in the cell, but when this cell matures this organizing center disappears. The structure of the microtubules remains stable but is different in the different compartments: Axonal and dendritic microtubules are not associated with any MTOC. At first they may be associated, but they dissociate when neurites are formed. Dendritic microtubules are shorter and have mixed polarity. Axonal microtubules contain stable segments that are unusually resistant to treatments that depolymerize microtubules in other cells. These short, stable microtubule segments serve to nucleate and organize axon microtubules, particularly during regeneration. The distances between microtubule filaments differ in dendrites (60nm) and in small-caliber axons without neurofilaments (20nm). 4.8. PROTEINS ASSOCIATED WITH MICROTUBULES There are proteins calledmicrotubule associated proteins (MAPs),which interact with microtubules to modulate their structure and function. Thus, theMAPsmore classic ones have a fundamentally stabilizing function thus inhibiting depolymerization and obtaining longer and less dynamic microtubules. On the other hand, other MAPs such ascatastrophicthey have a destabilizing function through their contact with the positive end of the microtubules. MAPs are proteins that co-purify with microtubules from neuronal tissue extracts. They also have other functions such as facilitating certain interactions with other molecules. Furthermore, there are not only neuron-specific MAPs, but also neuronal structure-specific ones. Examples are protein You, which is specific for axons, and proteinsMAP2A and MAP2B, which are specific to dendrites. In the image below we can see a series of proteins associated with microtubules, which are of different types, but share amicrotubule binding domain.If we look specifically at the proteins MAP2a and MAP2b, which are specific for dendrites, we can observe aprojection domainwhich gives these proteins a greater length compared to other MAPs. Interacting with a smaller protein in the case of axons (Tau) means that microtubules may be more clustered in axons than in axons. In dendrites, since the microtubules interact through MAP2 proteins, the distance between them is greater. We can see this in the electron microscopy images on the right. TAU PROTEIN Thetau proteinis a protein that has at least 6 slightly different isoforms produced by alternative splicing. This protein creates bridges between the different microtubules of the axon. When cells are transfected with the tau protein, an outgrowth of microtubule-containing extensions is induced. In addition, the phosphorylation of the Tau protein can affect its affinity for microtubules, its cellular localization or its functionality, thus having important implications in the health of the neuron. Different phosphorylations of the Tau protein are associated with multiple neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease or dementia with Lewi bodies. The Tau protein can have mutations, some of which can cause diseases such as familial frontotemporal dementia (FTD). 4.9. INTRACELLULAR TRANSPORT IN NEURONS As we already know, neurons are highly polarized cells that have their different compartments very separate from each other. Most macromolecules are synthesized in the soma, but are needed throughout the cell. In addition, some specific mRNAs must be transported to distal compartments in order to carry out local translation. All of this makes us think that different intracellular transport mechanisms are needed that can transport the different molecules, organelles and vesicles from the soma to the distal compartments of the neuron. This is achieved thanks to thetwo-way transport, which is based on molecular motors that use microtubules as pathways. This transport can be divided into anterograde and retrograde transport. - Anterograde transport - It is the transport that occurs from the negative end of the microtubule to the negative end. - It is mediated by the motors ofkinesin - Proteins, vesicles and organelles are transported from the soma to the axon terminal.  - Retrograde transport - It is the transport that occurs from the positive end of the microtubule to the positive end. - It is mediated by the motors ofdinner - It is the transport that allows the different organelles, endosomes, signaling molecules, etc. to return to the compartment of origin. KINESIN AND DINEIN MOTORS Molecular motors are multiprotein complexes capable of interacting with microtubules, producing conformational changes that allow eu movement over microtubules, and interacting with specific cargoes. Going a little deeper: - Thekinesin motorsthey are multimeric proteins that consist of a heteroteramer of 2 light chains and 2 heavy chains. Heavy chains bind to microtubules and light chains bind to cargo. - Thedynein motorsthey are multimeric proteins that contain light, medium and heavy chains, and that require a protein called dynactin to perform their function. Thedynactinis a complex of 11 proteins that is able to bind charges. KINESIN MOTORS The kinesin family is complex, and there are many types. Neuron-specific kinesins are theKIF5Aand theKIF5C, while KIF5B has a ubiquitous expression. Regarding cargo, we note that kinesin motors can transport a wide variety of molecules, such as vesicles, organelles such as mitochondria, mRNAs... The mitochondriaare transported byKIF1Ba (kinesin family 3) and the motors of KIF5. In addition, this transport capacity can be regulated in multiple ways, not only by the expression of neuron-specific kinesins and dyneins, but also by different processes, such as phosphorylations (in KIF5: PKA inhibits association with synaptic vesicles and GSK3 inhibits association with organelle membranes, and in KIF17: CAMKII induces release of vesicle- containing NR2B),action of GTPases or signaling through calcium. MESSENGER RNA TRANSPORT AND LOCAL TRANSLATION In different situations, due to external elements, a fast and specific translation may be necessary in certain compartments of the neuron,including localized regulation of the cytoskeleton. For example a stimulus that the growth cone receives, a large amount of actin is needed for the axon to grow. It can also be produced by electrical stimuli. In this situation with the gradual arrival of proteins is not enough, so the arrival of mRNAs for local synthesis of proteins is very important. mRNA sequence elements direct transport to distal neuronal processes by interacting with RNA-binding proteins (RBPs) that have a range of roles, including interaction with active transport motor proteins such as kinesins and the inhibition of mRNA translation during transport. mRNAs are transported as part of ribonucleoprotein (RNP) in a translationally inhibited state (e.g. ZBP1 (zip code binding protein 1) binds to b-actin mRNA in the nucleus and n 'inhibits translation). Activity can control mRNA transport (e.g. increase the number of granules carrying CAMKII, ZBP1 and Arc) 5. ACTIN MICROFILAMENTS 5.1. GENERALITIES The third of the elements that make up the cytoskeleton are themicrofilaments,which are about 8nm thick and are composed of actin. Theactinit is a protein conserved between species and very abundant in our body that can constitute 1-5% of the total protein in cells (up to 10% in muscle cells). In the case of neurons, microfilaments are important for the interaction between cytoskeleton and plasma membrane, as well as for giving shape and organization to surface structures. For example, when small ramifications form in the membrane to form dendrites or growth cones, the contribution of microfilaments at the beginning is essential. In addition, microfilaments also serve as a pathway for proteins called myosinsthey can transport organelles and molecules over short distances. 5.2. THE ACTIN As we have already said, microfilaments are made up of actin. Actin is a highly conserved protein that has different isoforms, some of them specific for certain functions. It is a 43kDa protein with 4 subdomains that make up 2 lobes. As for the isoforms, theβ and γ isoformsare those present in neurons. Actin is able to bind ATP, which it hydrolyzes to convert it into ADP. 5.3. STRUCTURE OF MICROFILAMENTS Actin in the globular state is in the statemonomeric formed by 4 subdomains. In the structure of microfilaments it is formed by two chains that create a double helix. Each of the strings is named after protofilamentEach actin molecule interacts with the successive actin molecule of the same protofilament, but also with the lateral protofilament. The existence of a polarity in the structure of the filaments will be important for their function. As in the case of microtubules, microfilaments have an end with a preference for growth and a region of preferential depolymerization. 5.4. ACTIN NUCLEATION As in the case of microtubules, nucleation Spontaneous nucleation:It occurs depending on the degree of concentration of soluble act. An initial nucleus of actin monomers is created and from there the microfilament begins to grow little by little. Formin-mediated nucleation:It occurs in regions close to the plasma membrane thanks to the formin complex, which is activated by Rho-GTP.  Arp2/3-mediated nucleation and branching:Apart from continuous nucleation and that which occurs in the plasma membrane, there can also be the formation of new actin filaments from the formation of branches on preformed microfilaments. This can happen thanks to the Arp2/3 complex, made up of proteins homologous to actin. From the central position of a preformed microfilament, this complex can create a new filament that maintains orientation and polarity. 5.5. ELONGATION OF MICROFILAMENTS The elongation of the microfilaments occurs at the positive end of the microfilament with the incorporation of monomeric actin bound to an ATP molecule. ATP is hydrolyzed within the microfilament, releasing inorganic phosphate, and ADP is released from the negative end of the microfilament. All this creates a general movement of the microfilament towards the positive side. 5.6. ACTIN CYCLE In the following image we can see theactin cycle.We see how ATP enters the positive end, which is hydrolyzed releasing ADP on the negative side. This also happens thanks to the action of proteins that interact with the actin filaments. For example, thecofilinis able to depolymerize regions enriched with ADP+actin. This causes the microfilament to fragment into pieces that will fragment at the same time into monomers, and each of the monomers will be attached to cofilin. These actin monomers with ADP will have the ability to return to the form of actin+ATP thanks to a protein calledporphyline. Porphilin favors the entry of ATP instead of ADP and the growth of the microfilament in the positive side thanks to nucleation mediated by Arp2/3 or formin. Thus, the cycle begins again. 5.7. PROTEINS ASSOCIATED WITH MICROFILAMENTS Not only cofilin and porfilin can interact with microfilaments; there is a large amount of proteins associated with microfilamentswhich have many different functions. Some participate in nucleation, others in interactions, in creating networks with a gel form, in providing contractility, etc. For example, the spectrinis a protein that stabilizes actin microfilaments with a morphology in the plasma membrane, or another example is thefimbrin,which allows filaments to be structured in a parallel shape in contralateral growth regions such as microvilli or filopodia. 5.8. REGULATION OF THE DYNAMICS OF THE MICROFILAMENTS It is possible that the process of actin polymerization occurs as a result of external signals, such as the arrival of a growth factor at the cell membrane. This, through receptors that bind GTP can trigger nucleation or branching processes through Arp2/3 complexes, which can produce the formation of filopodia or lamellopodia. Stress fibers can also form by similar mechanisms. This is quite important for axon growth. These external signals can activate mechanisms of aggregation and segregation, or of recycling and segmentation, thus intervening in many points that can promote or reduce the presence of actin fibers. 5.9. MYOSIN MOTORS IN THE TRANSPORT OF ORGANLES IN NEURONS As we have said previously, microfilaments are responsible for the mobility of organelles at short distances in certain regions of the neuron. This occurs thanks to themyosin motors.For example, we can have the case that certain organelles and charges need to arrive inside a dendritic spine, and the actin cytoskeleton is responsible for this. This cytoskeleton participates in different processes, such as the arrival of components inside dendritic spines, at presynaptic buttons, growth cone modulations, or even very specific modulations within the axon. In addition, apart from these transport functions, myosin motors and the actin cytoskeleton participate in processes such as the formation of new organelles, as is the case with the formation of mitochondria from mitochondrial fission. They also participate in the recycling of vesicles in the terminal button in the presynaptic part, or in the formation of vesicles from the Golgi apparatus. 6. DISTRIBUTION OF THE CYTOSKELETON IN NEURONAL STRUCTURES 6.1. GENERALITIES In the following image we can see a summary of the distribution of microtubules in a neuron. We observe that there are microtubules everywhere, but that the associated proteins are different (we observe Tau protein in the axon and MAP2 proteins in the dendrites). In addition, we also see that in the axon all the microtubules have the positive side oriented in the same direction, thus conferring a polarity. In contrast, dendrites have a mixed orientation or polarity, where half of the axons are oriented towards the soma and the other half towards the tip. In the following figure we see somewhat the same as in the previous one, with the exception that we can appreciate how the axon of the neuron has a very high amount of neurofilaments that confer structure. There is a greater presence of neurofilaments in axons than in dendrites. In addition, along the axonal tract it is common to find actin rings that add greater complexity to the structure of the different neuronal structures. We also find actin in the growth cone, dendritic fibers or dendritic spines. This makes us see that all types of cytoskeleton cooperate to maintain an optimal structure and functionality of the neuron and its structures. This also explains what we have already discussed that microtubules and dynein/kinesin motors are essential for long-distance transport, while short-distance transport relies on actin and myosin motors. 6.2. NEURONAL CYTOSKELETON IN THE GROWTH CONE In the specific case of the axonal growth cone, we can see how this complexity of the cytoskeleton is also present. When the developing growth cone is forming, we can see how the microtubules occupy the central part, while the outermost part, which is exploring where the cone should grow based on external signals, is formed by filopodia and lamellopodia formed by microfilaments. Then, this exploratory region of microfilaments is first formed and grows towards a specific region that presents an attractor element. If growth finally consolidates and stabilizes, it will be when the elements of the tubulin cytoskeleton begin to form, which will give more stability. It should be mentioned that when creating side branches the exact same thing happens. 6.3. NEURONAL CYTOSKELETON IN AXONAL FIBERS In axonal fibers the composition of the cytoskeleton is also very complex. Although they are enriched in neurofilaments, there has structures specific namesactin waves, actin rings and hot spots and trails,which are formed by actin and give specific and localized functions (he said nothing more). NEURONAL CYTOSKELETON IN THE INITIAL SEGMENT OF THE AXON In addition, all axons have a region at their beginning that represents a very important and useful structure for the compartmentalization and differentiation between the structures of the axon and the dendrite. This structure presents various elements of the cytoskeleton. On the one hand, it presents microtubules with specific post-translational modifications, but also disturbed actin filaments that are necessary to restrict diffusion between the cell body and the axon. 6.4. NEURONAL CYTOSKELETON IN THE GLUTAMATERGIC SYNAPSIS We find specific cytoskeleton in both the presynaptic and postsynaptic region. We observe how both regions are fundamentally enriched by the actin cytoskeleton which, depending on the degree of activity the cell must have, participates in the mobility of vesicles at the presynaptic end or of receptors at the postsynaptic end. theactinis the main pre- and post-synaptic cytoskeletal protein. Alterations in actin dynamics in dendritic spines have significant consequences for cognition. At the presynaptic level, the function of the cytoskeleton is to act as a scaffold to restrict the motility of the vesicles in the reserve pool (RP), leading by direct transfer the vesicles from the RP to the easy release pool (RRP) in the active zone. Actin is associated with short filaments of synapsin that are attached to vesicles. Phosphorylation of synapsin releases RP. In contrast, at the postsynaptic level, actin is enriched in the postsynaptic density (PSD) to anchor receptors. However, actin is present throughout the spine head. MF depolymerization disperses receptors, MFs are highly dynamic on spines, which is essential for synaptic plasticity. Actin contributes to the differential organization of different groups of receptors. Drebrin interacts with F-actin, reducing trademilling and modifying signaling responses. 7. NEURONAL POLARIZATION As we have already seen, the composition, orientation and structure of the cytoskeleton in different regions of the neuron is very different, so knowing how neurons can be so polarized is very important. When neurons begin to formin vitro, there is a first phase in which a first protrusion begins to appear in the body of the cell that in the future will be the neuron. At this point, there is no evidence that the protruding protrusion is already predefined as an axon or as a dendrite. Gradually more protuberances come out and everything is defined from different mechanisms. In the end, when the neuron is fully mature, we have microtubule structures released from the organizing center that are highly stable (much more in axons than in dendrites), and we also have maximum polarization. The initial stage is produced from several elements. At first we start from a basal situation (stage 2) in which we have an immature neuron with several small extensions called neurites. These neurites have a distribution of microtubules with a mixed orientation, but suddenly at one of the branches, the microtubules begin to organize to form the axon. This happens thanks to the participation of molecules such as TRIM46, which is able to stabilize the microtubules in a polarized way, achieving an absolute polarity where all the microtubules have the positive pole towards the distal end. Thus, we have an axon with an absolute polarity and dendrites that still have a mixed polarity. Some of the models to explain this polarization include two fundamental phenomena. On the one hand, a phenomenon occursglobal inhibition, where there are negative stimuli that make neurites less likely to develop. These global inhibition stimuli are combined with phenomenaof local activation, where a single neurite receives positive stimuli that cause it to grow and become the axon. Global inhibition allows the rest of the neurites to not become axons. Finally, it must be said that maintaining this polarization throughout the life of the cell is essential. For example, in Alzheimer's disease, the toxic β-amyloid peptide in contact with a mature, healthy neuron causes Tau protein not only to be found in the axon, but also to end up in the somatodendritic compartment, thus producing the characteristic aggregations of this disease that end up producing neuronal death.  Q4. REMODELING OF THE CYTOSKELETON OF MICROTUBULES DURING THE NEURONAL DEVELOPMENT. LEARNING ABOUT MITOSIS Microtubules (MT) are polymers of alpha and beta tubulin. They have an intrinsic polarity defined by the way in which the heterodimers of alpha and beta tubulin are assembled in the polymer constituting a positive and a negative end. Microtubules are organized in cells for essential processes: - Intracellular transport: they are generally involved in organizing the cytoplasmic arrangement of the organelles. - Segregation of chromosomes in cell division. To carry out these functions, the MTs are organized in different matrices or structures, which can be specific to the cellular stage and depend on the process in which they are participating (eg: mitotic spindle in dividing cells, axon in mature neurons, etc.). In mitosis, for example, the MTs are organized with a specific polarity: the ends (+) are joined to the chromosomes and the (-) to the pole of the mitotic spindle. The MTs are arranged in parallel. This parallel arrangement, in fact, is very similar between processes regardless of the general geometry of the microtubular matrix, because we also observe it in the axons. In the case of mature neurons, the axonal MTs are organized in parallel in a very packed form. The axonic and somatodendritic compartments in neurons differ in how MTs are organized. In general, in the axon all MTs have the (+) pole directed towards the end of the axon. However, in dendrites the polarity of the MTs is mixed, with part of the MT with the pole (+) towards the end of the dendrite and another part in the opposite direction. This difference in the organization of the MT cytoskeleton defines the identity of these compartments in neurons. This happens because motor proteins that transport charges along the neuron can read the specific polarity of the microtubules and transport charges selectively to the axon or dendrites. In this way, differences are established between the two compartments. There are different types of cargo that are transported by different classes of MT motor proteins, which can bedinner (they move the charge towards the end -) okinesins(most move charge towards the + extreme). MICROTUBULES ARE DYNAMIC POLYMERS Although they are very stable in neurons, MTs are not static. In fact, they are very dynamic. This is very important for its function. The dynamics of microtubules occurs mainly at the (+) end, where they grow. However, at this end a decrease (destabilization and depolymerization) can also occur, which we call catastrophe. MTs undergo constant transitions between growth and catastrophe, a phenomenon calleddynamic instability , which depends on the hydrolysis of GTP in the beta-tubulin molecules of the polymer. We can observe MT dynamism, for example, by marking MT binding proteins specific to the end (+) (EB proteins) in a mitotic spindle. it is observed that the MTs are constantly growing from the pole of the mitotic spindle to the chromosomes, which are aligning in the center. In the mitotic spindle, the dynamism of the MTs is essential to establish the connections with the kinetochores of the chromosomes and unite them to the two poles of the mitotic spindle. Many times these connections are incorrect, and the MT must disunite, depolymerize and re-unite again through polymerization. In addition, there are hundreds of dynamic MTs that leave the pole of the mitotic spindle and connect with the cell cortex allowing the spindle to be positioned and oriented correctly. This is important in the tissue context, because it is important that the cells divide in a specific direction to maintain the integrity of the tissue. In the neurons we also find MT dynamism. An example is the axon, the compartment with the most stable MTs. If we mark EB proteins, we observe constant growth of the ends (+) in the axon (they look like "comets") in the same direction, since in the axon they have a uniform polarity with the end (+) towards the end of the axon. Another example of MT dynamism in neurons is the growth cone of the axon. During the development of the SN, the end of the axon must find its way to connect with other neurons. Actin MFs in the cone cortex contribute to this process, but also dynamic MTs that grow and decrease exploring the surrounding area. The presence of gradients of molecules is detected by the growth cone and this allows a stabilization of the MTs in the face of attractive molecules, or their destabilization in the face of repulsive molecules. MTs, therefore, reinforce the direction in which the growth cone grows by reacting to extracellular signals. HOW ARE THE CONFIGURATIONS OF MICROTUBULES ORGANIZED? The fundamental mechanism by which cells mediate the organization of MT networks is controlling where and when MTs nucleate. MTs are not assembled randomly or spontaneously, but a highly controlled nucleation process occurs in specific locations of the cells called MTOCs (MT organizing centers). An activation of the nucleation process is required to start. A second important mechanism is the anchoring of the (-) end to structures such as MTOCs once they have nucleated and are growing. So the cells can control the general shape of the MT matrices or networks. γ-TUBULIN RING COMPLEX (γTURC) IN THE MTOC The main MTOC in cells is thecentrosome, constituted by two centrioles (mother and child) oriented perpendicularly and surrounded by a pericentriolar material. The mother centriole has associated proteins (appendages and satellites) that contribute to the organization of the pericentriolar material, which serves as the assembly site for the γTURC complexes in the area. TheγTURKis a protein complex that acts as a MT nucleator. These initiate the formation (nucleation) of MTs, constituting a molecular basis for the formation of new MTs. These complexes provide a template in which alpha-beta tubulin dimers can join to polymerize. The complex acts as a platform that simulates the (+) end of a MT. γTURC is composed of different subunits: - GCPs(gamma complex proteins). - Gamma tubulin:it is related to alpha and beta tubulins but it is not part of the MT, but of the nucleator. It forms the platform for the union of tubulin dimers. The γTURCs can also be anchored to the centrioles, to constitute a radial MT synthesis. MTs do not only grow at the level of the centrosome. There are many types of MTOC depending on the cell type. In some cell types, the surface of the Golgi apparatus can act as an MTOC. The nuclear surface can also act as MTOC in the case of muscle cells. It has recently been seen that mitochondria can also act as MTOCs in Drosophila spermatozoa. Therefore, cells can use a wide variety of structures, not just centrioles, as a basis for MT growth. Many people consider the centrioles very important for the organization of MT, because they allow the centrosome to form, but currently it has been seen that the essential function of the centrioles is the assembly of cilia. The cilia are formed in the centriole from MTs and are exposed on the surface. - theremulticiliated cellswhich can have a large number of cilia on the surface. These can be mobile, for example, in the trachea, transporting liquid and mucus on the cell surface through coordinated movements. - They also existprimary cilia. It is a single cilium formed from a centriole that plays an important role in signaling. They have a specialized membrane that continues with the plasma membrane, but between them there is a separation barrier which controls the composition of the ciliary membrane. There are receptors, for example, that are only present in the cilium membrane. The most relevant example of signaling through primary cilia is thevia Hedgehogin vertebrates. Ciliary membrane receptors receive extracellular signals and transduce them to the cell nucleus to regulate gene expression. In a cell that carries out the cell cycle, the microtubules leave radially from the MTOC (centrosomes). Along the interphase, the centrosomes are duplicated and the MTs are organized in the form of a bipolar mitotic spindle. Many cells, when they begin to differentiate, generate a primary cilium capable of receiving extracellular signals that will modify gene expression, a fact that will allow differentiation to continue and definitively exit the cell cycle. This is very important for development. Post-mitotic cells, like neurons, usually completely remodel the microtubule network and in many cases cases this will no longer depend on the centrosomes. Most of the cells in our organism are post-mitotic and do not organize MTs from the centrosome (many times they have even lost the centrosome). MICROTUBULES IN NEURONAL DIFFERENTIATION In recent years, interest has increased in understanding the transition from proliferative to post-mitotic (differentiated) cells and, in fact, neurodevelopment is a good model to study it. We observe in the image the cortical neuronal development model of the mouse. In early embryonic stages, there are many stem cells (radial glia) in the neuroepithelium proliferating in an expansive and symmetrical manner. In later stages, asymmetric divisions begin to occur that will on the one hand give rise to a progenitor cell and, on the other hand, to a daughter cell that will migrate to the surface and differentiate into a neuron (or glia). During the migration, the cell acquires a bipolar structure, so that one pole will become an axon and the other into dendrites. Therefore, different phases occur in cortical development: proliferation, migration and morphological differentiation. All these phases are very controlled by the microtubules and require their remodeling. Defects in the MT cytoskeleton have been associated with neurodevelopmental diseases For example, defects in MTs that affect the proliferation of neural progenitors have been associated withmicrocephaly. In microcephaly, although the development of the brain is relatively normal, it is much smaller in size (including the skull). It is believed that this is caused by the fact that the progenitors do not manage to generate a sufficient number of daughter cells to achieve a normal brain size. Another type of disease isLissencephaly, in which the brains have abnormal clefts caused by defects in neuronal migration. Mutations in proteins associated with microtubules that participate in these processes end up resulting in these types of defects. There are other diseases related to cognitive defects, motor defects and neurodegeneration that have also been related to defects at the MT level (disruption or dysfunction of the MT cytoskeleton). However, they are not so well characterized. RESEARCH LINES OF DR. JENS LUDERS I KNOW ABOUT MICROTUBULES IN NEURONAL PROLIFERATION It is believed that the formation of the bipolar mitotic spindle is caused by the combination of 3 mechanisms: - Nucleation by centrosome:is the main one - Nucleation mediated by chromatin: when the nuclear membrane breaks at the beginning of mitosis, the chromatin is exposed to the cytoplasm and can initiate the nucleation of the microtubules around the chromosomes through a pathway that depends on RanGTP. This pathway will result in the activation of γTURC-dependent nucleation and other assembly factors of the mitotic spindle. - Nucleation mediated by Augmina: MTs are nucleated on the surface of other MTs. Augmina is a protein complex that has been described as an amplification mechanism, then it joins the MTs and allows its number to rapidly increase from already existing MTs (branching nucleation). What happens if we interfere in any of these ways? The elimination of centrosomic nucleation usually results in microcephaly. Therefore, it is believed that this type of nucleation is not essential since, although the cells do not divide so efficiently, the other two ways can fulfill their function and form the mitotic spindle. There are mouse models in which chromatin-mediated nucleation has been inhibited. It has been seen that the inhibition of this pathway is lethal and the embryos do not develop. The same effect has been observed when making KO mice for the Augmina pathway. However, there is a problem with these results. In very early stages after fertilization, mouse embryos do not have centrioles, but human embryos do. Mouse centrioles are generated anew in later stages of embryonic development. This causes, by removing the Augmin pathway and the one mediated by chromatin in mouse embryos, there is no nucleation pathway present and these cannot develop, a fact that directly affects the aforementioned results. For this reason, Jens Luders' laboratory wanted to contribute to the knowledge of Augmina-dependent nucleation during mouse brain development. Studying augmin in specific stages of mouse brain development They generated miceConditional KO (floxed) for the Haus 6 gene, which codes for a central subunit of the augmina complex. We wanted to verify how important the augmin pathway was for the mitosis of neural progenitors in early stages of neurodevelopment. The conditional KO mice were crossed with mice that expressed the recombinase Cre specifically in the population of proliferative progenitors (apical progenitors) during neurodevelopment. The expression of Cre begins towards the embryonic stage E10.5, so that in these cells Cre can eliminate exon 1 of the Haus6 gene by cutting specific loxP sequences inserted around the exon. Next, different embryonic stages were analyzed following the effects of Haus6 deletion. The embryos did not survive, they died approximately at birth. It can be observed thatBrain development did not occur correctly in KO mice, because in tissue sections it is observed that there are no structures of the anterior brain (forebrain) in these animals. There is a severe disruption of the tissue already from early stages of development (E13.5). In an immunofluorescence against the apical progenitors (marking against pax6) and differentiated neurons (marking against βIII-tubulin) observed onelower tissue thickness in mice KO for Haus6,fact that indicates a development and growth of the tissue much less. To see if it was a defect in mitosis, immunofluorescence was performed against the mitotic marker p-H3. One was observedincrease of mitotic cells in the Haus6 KO condition,the majority in the subventricular region. Later it was seen thatin Haus6 KO they had mainly increased the cells in prometaphase state. This is consistent with a defect in the mitotic spindle, since the number of cells in the following stages of mitosis was already significantly lower in the KO. He then went deeper into the study of the mitotic spindle by means of a markergamma tubulin. In the KO mice, this was less marked and more fragmentedat the level of the poles of the mitotic spindle, a fact that allows us to deduce that the centrosomes do not maintain their integrity. Alpha tubulin was also marked and one was seen abnormal structure of the mitotic spindles themselves in the KO mice. What is the consequence of these defective mitotic spindles? At the cellular level it was observedin Haus6 KO mice a massive induction of p53 and apoptosis in progenitor cells(are the cell population where the KO was specifically done). So, if the disruption of mitosis caused apoptosis and neuronal death, could this explain the poor development of the brain in the mouse? p53 was knocked out in transgenic mice that had a conditional KO of Haus6 (floxed). They were crossed with mice that expressed Cre specifically in neuronal progenitor cells and the effect of the co-deletion was observed at different stages of neurodevelopment. With markers of apoptosis (cleaved caspase-3) it was observed that a KO of p53 rescued the neuronal death produced by the KO of Haus6. However, although certain growth and more cell proliferation is observed, the brains of mice with the co-deletion are still abnormal (they do not seem to be functional, there is a disorganization of the layers). In more advanced stages, the loss of tissue is more clearly seen, so that the elimination of p53 does not efficiently rescue the defects in neurodevelopment. In the double KO, an increase in mitotic cells was found in different parts of the forebrain, but we again see a greater increase at the prometaphase level, and a decrease in the later phases. This indicates that a KO of p53 prevents cell death, but every time the cells try to enter mitosis, they encounter defects in the mitotic spindle that will accumulate, preventing proliferation. An increase in DNA damage was found in the cells corresponding to the Haus6 KO (even with the co-deletion with p53 KO) due to chromosome breaks due to abnormal divisions (double-strand breaks detected with gamma-H2AX staining, phosphorylated histone in DNA repair processes). Next, the tissue organization was looked at in the double KOs and it was seen that the cellular organization had been lost, and that the progenitor cells (pax6+) were distributed in a dispersed manner, in the same way as the differentiated neurons. When marking gamma-tubulin, it was seen that in WT the centrosomes are aligned on the apical surface, while in the double KOs, the centrosomes were practically not visible. It was also seen that the polarity of the neural progenitors was lost in the double KOs (in WT condition they extend a prolongation that allows the migration of neurons in differentiation). A specific staining of the MTs (alpha-tubulin marking) showed a disruption and disorganization of the MTs themselves in the neural progenitors. Summary 1 Augmin is very important from mitotic spindle assembly and progression in apical progenitors mitotic defects after loss of augmin induce massive apoptosis apoptosis is apical progenitors lacking augmin is rescued by p53 co-deletion p53 co-deletion does not rescue brain development continued proliferation of Hays6 p53 double KO progenitors leads to aberrant tissue morphogenesis augmin may have a role in apical progenitors during interphase I KNOW ABOUT MICROTUBULES IN DIFFERENTIATING NEURONS Once the neural stem cells divide asymmetrically, the immature neurons will begin the migration process in which they will undergo a morphological differentiation in which they will develop somatic processes, one of which will give rise to the axon and the rest to dendrites. This differentiation in neuronal polarity requires reorganization of the MT cytoskeleton to define the somatodendritic (with mixed MT polarity) and axonal (with uniform MT polarity) compartments. As previously mentioned, when the cells differentiate, in most cases the centrosomes will no longer be a main MTOC. Therefore, how do these cells generate MT without a centrosome and how do they organize them? One of the first factors that was analyzed was the activity of the centrosome in hippocampal neurons in very early phases of differentiation. As the days pass in vitro, the gamma-tubulin signal weakens and therefore, there is a loss of activity at the level of the centrosome. In the case of pericentrin (marker of centrioles) constant levels are observed, so thatthe centrosome is not lost as such, but loses its nucleating capacity due to loss of gamma- tubulin. Through WB, it was observed that the concentration ofgamma- tubulin was not reduced in the neurons, but it was no longer located in the centrosomes. This is corroborated with a reduction of NEDD1, whose function is to bind gamma-tubulin to the centrosomes. A knock down was made for gamma-tubulin and the microtubules were analyzed by detecting proteins at their (+) ends. In the KD there is a clear decrease in MTs. Therefore, MT nucleation in differentiated neurons depends on γTURC and the presence of gamma tubulin, but where do MTs nucleate if not in the centrosome? By detection of alpha-tubulin, it was seen that the organization and arrangement of MT in KD by gamma tubulin was also there altered This suggests that gamma-tubulin not only promotes nucleation but also the organization of MTs. When describing the pathway of augmin in the nucleation of MT during mitosis in the assembly of the mitotic spindles, we wanted to see if this pathway was also a mechanism used in differentiated neurons. In a recent test where MTs were marked in red and their ends (+) in green, it was shown that MTs nucleate from the surface of existing MTs, and this quickly allowed the MT network to be amplified generating large MT bundles. This supports the augmina pathway as a mechanism, because it is independent of the central structure and can occur far from the neuronal body (as in the axon). In neurons in culture, by means of a WB with antibody against Haus6, it was observed that the augmin complex was present both in embryonic and postnatal stages. Through immunoprecipitation it was seen that this complex interacted with gamma-tubulin. Next, a KD of Haus1 (subunit of the augmin complex) was made in neurons in culture and a similar phenotype was observed to the one that occurred in a KD of gamma-tubulin. When monitoring the movement of the ends (+) of the MTs, it was seen that in a KD of the augmin complex, the MTs could move in the opposite direction to the normal one in the axon (abnormal polarity in some MTs). This allowed us to elucidate that the augmina complex controls, through the mechanism ofbranching, that the polarity of the MTs is maintained, because the new MT will have the same polarity as the MT to which the augmina complex is attached. Therefore, this type of nucleation ensures that the nucleated MTs have the same polarity as those from which they have been synthesized. To demonstrate this mechanism in a different way, ectopic nucleation (independent of augmin) was induced by means of the overexpression of an activation nucleator. By overexpressing this nucleation activator, incorrectly oriented MTs are observed. As we know, polarity is important for the transport of charges by motor proteins. When making a KD of gamma-tubulin, the transport is altered and there are very few charge movements. At the level of the morphology of the neurons, a KD of gamma-tubulin caused less development at the level of extensions, fewer ramifications and shorter ones than in the control condition. Therefore, inhibiting nucleation in neurons interferes with axonal transport and with the growth of extensions andbranchingof the MTs. Regulation of MT polarity by augmina-γTURC in axons It is believed that in the axon there are MTs that are nucleated from other MTs. This requires the augmin complex, which in turn recruits the γTURC complex to the surface of MTs to nucleate branches of new MTs. This ensures that all MTs in the axon maintain their uniform polarity, required by charge transport, growth andbranchingin the axon When augmin is lost, γTURC nucleation continues but in an uncontrolled and random manner that will occasionally give rise to MT with opposite polarity. When activating ectopic nucleation (promoting unregulated γTURC activation), even with the presence of augmin, some MTs will nucleate without the augmin complex in the opposite direction. Recent data There are unpublished data that show that the augmin complexes are not only acting in axons, but also in dendrites. A decrease in dendrites is observed when performing a KD for subunits of the augmin complex. It was seen that in a KD of augmin there were no significant changes in the dendritic polarity of the MTs, because as we know, in these there is a mixed polarity. What was found is a reduction in the number of ends (+) in growth, since most of the nucleating activities are inhibited when making the KD of augmin. So, why does augmin act in dendrites, if they have a mixed polarity? The reason is that the MTs in dendrites are not organized as individual MTs, but rather constitute bundles of MTs, each with a polarity. Therefore, it is believed that the augmin complexes are essential to maintain MT configurations in each dendritic MT bundle. It is now known that MTs, both in dendrites and in axons, constitute bundles, for the polarity and robustness of which the action of the augmin complex is necessary. This is a way in neurons to maintain MT networks in the absence of nucleation by centrosomes. This is very useful, because constant local nucleation is necessary in regions very distant from the neuron's soma (axon, dendrites...). Summary 2 Neurons employ decentralized non-centrosomal nucleation Nucleation is mediated by augmin, from the surface of other Mts. Augmin-mediated nucleation controls MT polarity in axons Augmin-mediated nucleation is used throughout all neuronal compartments Augmin-mediated nucleation is required for proper microtubule organization and function. Disruption of augmin-mediated nucleation impairs both transport and morphological differentiation.  T5. INTRACELLULAR SIGNALING IN THE NEURON 1. INTRODUCTION: CONCEPT The 3 main protagonists of intracellular signaling are theextracellular signal, thereceptor proteinand themtarget proteinswhich will give a temporary or permanent cellular response. Between the receptor protein and the target protein there is a whole series ofintracellular signaling proteinsthat what they do is translate the signal so that the cell responds to the signal through the target proteins. Target proteins can be of different types, such as metabolic enzymes, cytoskeleton proteins or gene regulation proteins. All this leads us to the fact that different responses can be produced in relation to a signal. 2. GENERALITIES 2.1 CHEMICAL SIGNALING MOLECULES The molecules that will initiate the signaling can be: Molecules impermeable to the cell→ when they are released they bind to transmembrane receptors of the target cell. This leads to the activation of the receptors and the transmission of the signal. Ex. Neurotransmitters, peptide hormones and trophic factors. Cell permeable molecules→ they will pass inside the cytoplasm of the receiving neuron, there they will find their receptors. Ex. Steroid hormones, thyroid hormones and retinoids. Molecules associated with the cell→ the two cells must be very close together for contact to occur between the molecule and its receptor. Ex. integrins and NCAM. 2.2 RECEIVERS There are different types of receivers: Receptors linked to canals→ a conformational change occurs in the receptor that allows the passage of ions. Ex. Ionotropic receptors of neurotransmitters. Enzyme-linked receptors→ the conformation of the receptor is produced, giving rise to the activation of an enzyme found inside the cytoplasm. Ex. Metabotropic receptors, receptors for neurotrophins. G protein-coupled receptors→ when the signal binds, the receptor attracts the G protein which is activated giving rise to a whole cascade of intracellular signaling. Ex. Metabotropic receptors, β-adrenergic receptor, metabotropic receptor for glutamate, odoriferous substance receptors, mAchR receptors. Intracellular receptors→ they are those whose ligand can easily cross the plasma membrane. RECEPTORS COUPLED TO G PROTEINS G proteins can be of two types: Monomeric→ formed by a single polypeptide chain, like RAS. trimeric→ formed by 3 subunits (α, β, γ). When the receptor is inactive, it is separated from the G protein. When activated, a conformational change occurs that allows it to come into contact with the G protein. The binding of the receptor to the G protein causes it to become active by binding GTP. Once the G protein is activated, it can interact with other proteins and activate them. EFFECTIVE PATHWAYS ASSOCIATED WITH G PROTEINS There are different isoforms of the heterotrimeric G proteins, so that the signal that will be triggered from them can be positive or negative: Gs. It activates adenylate cyclase and there is an increase in protein phosphorylation Gwhat. It activates phospholipase C and there is an increase in protein phosphorylation and the activation of Ca-fixing proteins2+. Gi. It inhibits adenylate cyclase and there is a decrease in protein phosphorylation. SECOND MESSENGERS Thesecond messengersare essential asactivators and regulators of many protein kinases and phosphatases: Ca 2+, cyclic nucleotides, DAG and IP3. 3. SIGNALING INDUCED BY PHOSPHORYLATION/DESPHOSPHORYLATION 3.1 PHOSPHORYLATION Thephosphorylationof proteins is a very widespread control mechanism. It is important for: Theactivation and inhibition of enzymes Theassembly of multiprotein complexes Some examples of regulation by phosphorylation are: Increase or decrease the biological activity of an enzyme. Help move proteins between subcellular compartments. Allow interactions between proteins. Mark proteins to be degraded. Phosphorylation is just oneenzymatic reaction in which the phosphate group is added(P.O4=)terminal of ATP to a protein. It's usually a processreversible which is carried out bykinasesyphosphatases. The phosphorylation/dephosphorylation system is a very complex signaling system due to: 1/3 of the proteins present in a typical mammalian cell have a phosphate group covalently attached. That is, this system includes a large number of proteins as target molecules. There is a large number of kinases and phosphatases. In addition, some are very general while others are very specific. Proteins can be phosphorylated by more than one kinase and a kinase can phosphorylate a protein at more than one site. The phosphorylation is able to control the enzymatic activity due to that causeconformational changes in the protein. This results in a protein having two conformations and, therefore, two different protein activities. protein phosphorylation will act as a molecular switch that allows us to have oneprotein enzymatically active or inactive. However, we must keep in mind that each phosphorylation can havedifferent effectson protein activity, can cause aincrease in activity, one decreaseeitherhave no effect. The targets of the kinases are the -OH groups of the amino acids:serina, threonine,tyrosineand histidine (only prokaryotes). Kinases are classified according to what they phosphorylate:serine/threonine kinaseortyrosine kinase. The advantages provided by the phosphorylation and dephosphorylation of proteins are: 1. Speed (seconds) 2. No synthesis or degradation of new proteins is required 3. It is easily reversible. Proteins that can be phosphorylated are: Enzymes Cellular receptors Signaling molecules Structural proteins Ionic channels Protein kinases and phosphatases are activated from extracellular signals. The signals join their membrane receptors, which activate proteins that will finally activate protein kinases and phosphatases. We can even have interconnected networksof signal amplifications. Different ligands bind to several receptors, which involves the activation of different kinases, giving rise to these networks. Specifically, kinases canbe activated by : Hormones directly through the tyr-kinase Through a cascade of kinases Growth factors receptor (phosphorylation 2a) Light Hormones indirectly through the Neurotransmitters Cytokines, etc. generation of secondary messengers 3.2 CLASSIFICATION OF KINASES IN VERTEBRATES As we have mentioned before, kinases are classified according to the function they phosphorylate, so we have: serine/threonine kinasesytyrosine kinases. PROTEIN-SERINE/THREONINE KINASES We classify them according to whether they aredependent or independent of second messengers: Protein kinases dependent on second messengers : ▪ Cyclic nucleotide (cAMP, cGMP) regulated:PKA,PKG ▪ Diacylglycerol (DAG) regulated:PKC ▪ Calcium/calmodulin regulated:CAMK Protein kinases independent of second messengers : MAP kinasescascade and target kinases: Raf, ▪ P21 activated kinases (PAK) MEKs kinases, MEKs, SEKs, ERKs, JNKs, SAPKs, ▪ Kinases involved in the organization of the RSKs cytoskeleton and development: ROCK1/Rho kinase ▪ Cyclin dependent kinases (CDKs) and CDK ▪ Transmembrane receptor protein serine/ regulating kinases: cdc2 family, cdc2-2-related threonine kinases: TGFβ receptor protein protein kinases, CAK, CAK kinase kinases ▪ G protein-coupled receptor kinases: GRK2, ▪ Casein kinases: CK1, CK2 GRK3, GRK5, GRK6 PKA – PROTEIN KINASE A Protein kinase A (PKA) is a protein dependent oncAMP. In suinactive status form onetetramercomposed of2 catalytic subunitsy2 regulatory subunits. As long as the catalytic subunits are attached to the regulatory layers, they will not have kinase activity. However,the binding of cAMP to regulatory subunitsproduces a conformational change in these subunits that leads to therelease of catalytic subunitsand consequently activation.