Lesson 3 - Cytoskeleton and Cell Junctions 2023-2024 PDF

CELL BIOLOGY AND HUMAN GENETICS Ve más allá Academic Year 2023-2024 DAVID BALLESTEROS PLAZA Department of Pre-clinical Dentistry (Building A) E-mail address: davidalberto.ballesteros @universidadeuropea.es Ve más allá Academic Year 2023-2024 LESSON 3 CYTOSKELETON AND CELL JUNCTIONS INTRODUCTION MICR...

CELL BIOLOGY AND HUMAN GENETICS Ve más allá Academic Year 2023-2024 DAVID BALLESTEROS PLAZA Department of Pre-clinical Dentistry (Building A) E-mail address: davidalberto.ballesteros @universidadeuropea.es Ve más allá Academic Year 2023-2024 LESSON 3 CYTOSKELETON AND CELL JUNCTIONS INTRODUCTION MICROTUBULES MICROFILAMENTS INTERMEDIATE FILAMENTS CELL JUNCTIONS 3 MICROTUBULES MICROFILAMENTS INTERMEDIATE FILAMENTS 4 4 INTRODUCTION The cytoskeleton is a dynamic network of protein filaments extending throughout the cytoplasm. The cytoskeleton is critical for many essential cell functions : cell division, cell movement, cell growth, differentiation... The main functions of the cytoskeleton are :  To provide a structural framework for the cell and support the large volume of cytoplasm (especially in animal cells that have no cell wall).  To serve as a scaffold that determines the position of organelles.  To facilitate the internal transport of organelles or other structures (including chromosomes during cell division).  To allow cell movements (cell migration), cell contraction, or changes in cell shape. 5 CYTOSKELETON COMPONENTS IN EUKARYOTIC CELL The cytoskeleton is composed of proteins that assemble through non-covalent interactions to form long filament structures. Three types of structures can distinguished based on their diameter, protein component and subunit arrangement :  MICROTUBULES (22-25 nm) hollow cylinders made of Tubulin  INTERMEDIATE FILAMENTS (10 nm) show a rope-like structure and are composed of fibrous proteins (keratin, vimentin...)  ACTIN FILAMENTS OR MICROFILAMENTS (5-9 nm) two stranded helycal polymers of Actin 6 CELLULAR DISTRIBUTION OF THE CYSTOSKELETON Each of the three cytoskeleton components show a different distribution in the cell. a) b) MICROTUBULES a) MICROTUBULES Centrosome c) MICROFILAMENTS INTERMEDIATE FILAMENTS radiate from the microtubule-organizing center (MTOC) or b) MICROFILAMENTS show a scattered distribution in the cytosol, but are especially concentrated just beneath the plasma membrane. c) INTERMEDIATE FILAMENTS extend accross the cytosol forming a framework that attaches to the plasma membrane and provides mechanical strength to the cell. 7 8 DIFERENCES COMPOSITION MICROTUBULES MICROFILAMENTS INTERMEDIATE FILAMENTS TUBULIN (globular) ACTIN (globular) KERATIN VIMENTIN NEUROFILAMENTS LAMINA….. POLARITY YES YES NO DYNAMIC INSTABILITY YES YES NO YES (GTP) YES (ATP) NO KINESIN - DINEIN MIOSIN NO ENERGETIC MOLECULE AS A REGULATOR MOTOR PROTEINS FUNCTION -Cell shape -Intracelular arrangement. -Cellular transport -Cillia and flagella -Mechanical support -Movement -Mechanical stability -¿??? 9 MICROTUBULES 10 MICROTUBULES Cells contain 2 types of microtubule populations:  UNSTABLE MICROTUBULES, short-lived and very dynamic. They form the Mitotic Spindle and mediate changes in cell shape and organelle movements  STABLE MICROTUBULES, long- lived and non dynamic Present in Cilia, Flagella and Centrioles Mitotic Spindle Flagellum Centrioles 11 STRUCTURE OF MICROTUBULES A microtubule is a polymer of globular tubulin subunits arranged in a hollow cylindrical tube. 13 Each microtubule subunit is a heterodimer of α - Tubulin and β-Tubulin. The tubulin subunits are aligned forming end to end protofilaments that pack side by side to create the wall of the microtubule. Each microtubule contains 13 protofilaments. Tubulins are very similar in all animal species and together with Histones are the most evolutionary conserved proteins. 12 Each protofilament has a structural POLARITY, with α-tubulin exposed at one end and β-tubulin at the other end. This polarity is the same for all the protofilaments thus confering a STRUCTURAL POLARITY to the whole microtubule. Diameter: 25 nm Length: variable (1- >100 μm) 13 MICROTUBULE DYNAMICS Microtubules are dynamic structures that go through continuous assembly and disassembly processes and are constantly growing and shrinking. This behavior is known as DYNAMIC INSTABILITY. In addition to their structural polarity, microtubule display GROWTH POLARITY because tubulin dimers are added preferentially at one end, designated the (+) end (fast growing end) , and are lost preferentially from the other end, the (-) end (non-growing or slow growing end). The (+) end is the one where the β-tubulin monomers are exposed. The (-) end is the one where the α-tubulin monomers are exposed. In a cell the (-) ends of all microtubules are found around a microtubule organizing center or MTOC (the centrosome for example) and radiate from there to the cell periphery. 14 MICROTUBULE ASSEMBLY AND DISASSEMBLY 1 αβ-tubulin dimers bind head to tail to form short protofilaments. 2 These proteofilaments associate laterally into more stable curved sheets. Eventually sheets made up of 13 protofilaments wrap around forming a short hollow cylinder. The microtubule then grows by the addition of new tubulin dimers to the ends of the protofilaments. 3 The free tubulin dimers have GTP bound to both tubulin monomers. However, shortly after the tubulin dimer is added to the growing microtubule, GTP in the βtubulin monomer is hydrolized to GDP due to the intrinsic GTPase activity of tubulin. 4 GTP hydrolysis weakens the binding affinity of tubulin dimers for each other leading to disassembly or depolimerization of the GDP-bound dimers. 15 The Dynamic Instability Model  Only microtubules with (+) ends associated with GTP-tubulin are stable and can prime tubulin polymerization.  MTs with bound GDP-tubulin depolymerize more rapidly and can disappear in 1 min.  At high concentrations of non-polymerized GTP-tubulin, the rate of tubulin assembly is faster than the rate of GTP hydrolysis. Therefore the MT grows.  At low concentrations of non-polymerized GTP-tubulin, the rate of tubulin assembly decreases and the GTP hydrolysis rate is higher. An unstable GDP cap is formed, the protofilaments spring apart and tubulin subunits are released. The MT shortens. 16 Microtubule disassembly and GTPase activity α-tubulin  non-hydrolyzable GTP β-tubulin  GTP is hydrolyzed to GDP 17 Both ends of the microtubule have the ability to grow, but they will do it at very different rates. Tubulin polimerization rate at the (+) end is 2-3 times higher than at the (-) end. The concentration of free tubulin dimers determines whether a microtubule will grow or shrink. The Critical Tubulin Concentration (Cc) is the concentration at which microtubule length is constant. If the tubulin dimer concentration is higher than Cc, polymerization will be favored and the microtubule will grow. If the tubulin dimer concentration is lower than Cc, depolymerization will be favored and the microtubule will shrink. The concentration of tubulin in the cytoplasm depends on the rate of synthesis and degradation but varies also with polymerization or depolymerization events. (-) (+) [Tubulin] = Crical Concentration (Cc) (-) (+) [Tubulin] > Cc Polymerization (-) [Tubulin] < Cc (+) Depolymerization 18 Dynamic Instability Model. Elongation and shrinkage of MTs At Tubulin dimer concentrations close to Cc, some microtubules will grow and others will shrink 19 MICROTUBULE-ASSOCIATED PROTEINS (MAPs) They bind specifically to microtubules and modulate their stability of and their association with other cell structures. There are two types of MAPs :  PROTEINS THAT STABILIZE MICROTUBULES : they bind to the negatively charged Cterminal part of Tubulin and stabilize the outer wall of a microtubule. They can increase the growth rate of microtubules or suppress microtubule catastrophe. They are cell-specific. Some are present in neurons : MAP1A, MAP1B, TAU (in axons and dendrites) and MAP2 (only present in dendrites). Others are not present in neurons : MAP4.  PROTEINS THAT DESTABILIZE MICROTUBULES : they may sever intact cytosolic microtubules through an ATP-dependent process by breaking-up internal bonds between tubulin dimers (Katanin ) or they may promote disassembly of tubulin dimers at the (+) end (Catastrophin) 20 Regulation The binding of MAPs to Microtubules is inhibited by PHOSPHORYLATION. Therefore, phosphorylation of MAPs favors microtubule disassembly. + MAPs 21 Tauopathies Neurodegenerative disorders associated to defects in the normal functioning of the microtubule associated protein TAU.  Alzheimer´s Disease : TAU is Hyperphosphorylated Hyperphosphorylated Tau aggregates into Neurofibrillary Tangles and sequesters normal Tau and other MAPs leading to severe microtubule instability. This causes neuronal death. Beta-amyloid plaques Neurofibrillary Tangles 22 23 VESICULAR TRANSPORT THROUGH MICROTUBULES Vesicles can be transported by MOTOR PROTEINS like Kinesin and Dinein moving along microtubules. The movement can be towards the (+) end (anterograde) or towards the (-) end (retrograde) of microtubules.  Kinesins mediate the anterograde transport  Dineins mediate the retrograde transport The type of receptors present on the vesicle surface will determine in what direction it will be transported. Vesicle transport is particularly important in neurons where molecules synthesized in the cell body or even mitochondria must be delivered to the axon terminal. Axonal transport occurs in both directions (up and down the axon) , is fast (up to 400 mm/day), and relies on kinesin, dinein and microtubules. http://www.youtube.com/watch?v=kOeJwQ0OXc4&feature=related 24 DRUGS THAT DISRUPT MICROTUBULE DYNAMICS  COLCHICINE interferes with polymerization because it binds tightly to tubulin dimers and prevents their assembly into microtubules. As a result, microtubules will disassemble.  TAXOL is a microtubule stabilizing drug that prevents tubulin depolymerization and alters microtubule dynamics (microtubules can grow but not shrink). It is especially toxic for activelly dividing cells like tumor cells and it is used for the treatment of ovarian, breast and lung tumors. These drugs alter the steady-state balance of unstable microtubules but they do not affect stable microtubule populations (Cilia, Flagella, Centrioles). They both inhibit mitosis and chromosome segregation. 25 FUNCTIONS OF UNSTABLE MICROTUBULES 1. MAINTAIN THE CELL SHAPE 2. CELLULAR TRANSPORT. Axonal transport The oriented microtubules in the axon serve as tracks for the directional transport 3. MITOTIC SPINDLE FORMATION, which will distribute chromosomes between daughter cells when the cell enters mitosis. 26 STABLE MICROTUBULES CILIA AND FLAGELLA CENTRIOLES 27 CILIA AND FLAGELLA  They are thin and flexible proyections of the cell.  Cilia and Flagella have essentially the same structure but cilia are short (few micrometers) and abundant while flagella are long (e.g. more than 2 mm in an insect sperm) and scarce.  Function: locomotion of free cells (sperm) or movement of fluids along the cell surface (respiratory epithelia).  Structure : 2 main regions Axoneme: central bundle of microtubules Basal body: point of attachment to the cell. 28 AXONEME  The axoneme is formed by 9 doublet MT surrounding a central pair of singlet MT (9+2 arrangement).  Each doublet consists of A and B microtubules (A is complete: 13 protofilaments; B is incomplete: 11 protofilaments) with the (+) end located at the distal end of the axoneme.  The outer MT doublets are connected to the central pair by radial spokes and to each other by a protein called Nexin.  Permanently attached to each A tubule of the doublet are inner-arm and outer-arm dyneins, that drive the movement of cilia and flagella. 29 BASAL BODY It is the growing point of cilia and flagella, where the (-) end of axoneme microtubules are oriented. It has the same structure as centrioles : 9 triplets of microtubules tilted towards the central axis. 30 Differences between cilia and flagella ① Type of movement : Cilia show a pendular or whiping movement, with an effective stroke followed by recovery stroke to initiate a new movement. They sweep materials across tissues. Flagella show a waving movement that propels cells forward. ② Lenght: Cilia are shorter than flagella. ③ Number : Cilia are present in high numbers in each cell while flagella are scarce (1 or 2) FLAGELLA CILIA 106/µm2 31 CENTRIOLES  Two cylindrical structures located at the center of the centrosome.  Exclusive of animal cells.  In interphase they are perpendicularly oriented and constitute the DIPLOSOME.  Together with the pericentriolar (PC) matrix they form the Centrosome  Each centriole is formed by 9 triplets of microtubules, tilted towards the central axis of the structure.  Each triplet is attached to the adjacent triplet by the NEXIN protein.  Before entering mitosis cells must duplicate their centrioles. Each centriole gives rise to a new centriole so that once mitosis is complete each daughter cell gets a diplosome.  They are involved in the formation of the mitotic spindle but they are not essential (e.g. plants do not have centrioles but form mitotic spindles). C C B A diplosome 32 THE CENTROSOME  The centrosome functions as a Microtubule Organizing Center (MTOC).  It is made up of pericentriolar material (PCM) and sometimes, but not always, it contains 2 centrioles oriented perpendicular to each other.  The (-) ends of cytosolic microtubules are immersed in the pericentriolar matrix but they do not contact the centrioles.  A gamma-tubulin ring (γTuRc) is found at the (-) end of microtubules , but the (+) ends are free. 33 MICROFILAMENTS 34 MICROFILAMENTS Microfilaments are the thinnest filaments in the cytoskeleton (5-9 nm) They are two stranded helical polymers of ACTIN Actin is the most abundant intracellular protein in eukaryotes Microfilaments show structural and growing polarity : (+) end , fast growing, and (-) end, slow growing.  The rate of polymerization and depolymerization is very high.  They are usually shorter and more flexible tan microtubules.     Actin F-ADP Actin G-ATP 35 ACTIN  Actin can be found as a free globular monomer (G-ACTIN) or it can form fibrous polymers (F-ACTIN).  Free Actin monomers are bound to ATP, but ATP is hydrolyzed to ADP shortly after it is incorporated into the filament.  Hydrolysis of ATP reduces the binding strength between actin monomers and leads to depolymerization. G-Actin F-Actin 36  The three dimensional structure of globular G-Actin shows two lobes separated by a deep cleft or groove where the ATP molecule sits.  Two ends or sides can be distinguished on Actin : the side with the ATP cleft and the opposite side.  At the (-) end of the filament Actin subunits are oriented with their ATP cleft pointing outwards. At the (+) end of the polimer, Actin subunits are oriented with their ATP cleft in contact with the preceeeding Actin unit and the opposite side facing outwards. (-) end (+) end (growing end) 37 POLYMERIZATION OF ACTIN MICROFILAMENTS 1) NUCLEATION : formation of small aggregates of Actin. It is the rate-limiting step in the formation of an actin polymer. 2) ELONGATION : the small initial nucleus elongates by addition of new actin units to both ends. The (+) end grows faster. 3) HYDROLYSIS OF ACTIN-BOUND ATP AND STABILIZATION : a steady state is reached at which the rate of addition of new subunits to the filament ends exactly balances the rate of subunit dissociation. The filament length does not change. 38 ACTIN FILAMENT TREADMILLING  ATP-actin monomers are added rapidly to the (+) end of the microfilament and the ATP is hydrolyzed to ADP after polymerization. The ADP-actin is less tightly bound and can dissociate from the (-) end.  This cycle is called TREADMILLING. It creates a flow of actin monomers from the (+) end to the (-) end of the filament.  Theadmilling illustrates the dynamic behavior of actin filaments. 39 ACTIN FILAMENT TREADMILLING 40 ACTIN-BINDING PROTEINS The formation and stability of actin filaments in the cytosol is controlled by different actin-binding proteins  PROTEINS THAT PROMOTE POLYMERIZATION: PROFILIN  PROTEINS THAT PREVENT POLYMERIZATION: THYMOSIN  SEVERING PROTEINS : GELSOLIN AND SEVERIN  CAPPING PROTEINS : CapZ AND TROPOMODULIN 41 REGULATION OF POLYMERIZATION-DEPOLYMERIZATION Actin polymerization is regulated by proteins that bind free actin monomers (Gactin). These proteins either promote or inhibit actin polymerization. PROTEINS THAT PROMOTE POLYMERIZATION: PROFILIN : it forms a complex with G-actin-ATP that contributes to monomer aggregation at the (+) end. It is a nucleotide-exchange factor. PROTEINS THAT INHIBIT POLYMERIZATION: THYMOSIN : it binds to G-actin-ATP, sequesters it and prevents its binding to the (+) end of the filament. 42 SEVERING PROTEINS Another group of proteins control the length of actin filaments by breaking them into shorter fragments. These proteins stabilize a conformational change in the actin subunit to which they bind and create a small gap between neighboring subunits and, eventually, a break. These proteins are GELSOLIN and COFILIN. After breaking a filament , the severing protein remains bound at the (+) end of one of the resulting fragments, where it prevents the addition or exchange of actin subunits (FILAMENT CAPPING). 43 CAPPING PROTEINS They bind to actin filament ends and stabilize them.  CapZ: binds to the (+) ends of actin filaments and prevents addition or loss of actin subunits at that end.  TROPOMODULIN: Caps the (-) ends of actin filaments CapZ An actin filament that is capped at both ends is effectively stabilized, undergoing neither addition nor loss of subunits. Such capped filaments are needed in places where the cytoskeleton organization does not change, as in a muscle sarcomere or in the erythrocyte membrane. 44 DIFFERENT ACTIN ARRAYS IN A CELL 45 ORGANIZATION OF ACTIN FILAMENTS Actin filaments filaments are assembled to form two types of stable structures : ACTIN BUNDLES ACTIN NETWORKS Both structures require ACTIN CROSS-LINKING PROTEINS to link one filament to another in different ways. The filament pattern created by this proteins is determined by their size and shape. Actin-Bundling proteins : FIMBRIN, α-ACTININ Network-forming proteins : FILAMIN 46 47 ASSOCIATION OF ACTIN FILAMENTS WITH THE PLASMA MEMBRANE Actin filaments are highly concentrated in the periphery of the cell where they form a structural network attached to the cell membrane called the CELL CORTEX. The cell cortex determines the cell shape and is important for cell motility. The cell cortex is particularly important in ERYTHROCYTES that lack other cytoskeletal components. The main component of erythrocytes cell cortex is SPECTRIN, a tetrameric protein with two actin-binding domains, that forms a two dimensional web held together by short ACTIN filaments and linked to the plasma membrane by peripheral membrane proteins like ANKYRIN. These peripheral proteins are bound to transmembrane proteins such as Glycophorin and Band 3. This structure determines the flexibility and shape of red blood cells. Mutations in genes coding for some of these proteins have been linked to hemolytic anemia. ABD SPECTRIN Actin-Binding Domain 48 MICROFILAMENT FUNCTIONS 1- DEFINING AND CHANGING THE CELL SHAPE : For example : changes in platelet shape during blood clotting. Resting platelets have a biconcave, disk-like shape Following activation by clotting agents platelets extend numerous FILOPODIA (thin cellular projections) Finally, platelets spread out and form LAMELLIPODIA (sheet-like extensions) These changes in morphology are the result of complex rearrangements in the actin cytoskeleton that is cross-linked to the plasma membrane. 49 2- FACILITATING CELL MOTILITY (CELL CRAWLING) AND MUSCLE CONTRACTION: 1)Extension of the cell membrane and formation of a lamellipodium. 2) Polymerization of actin filaments and further protrusion of the lamellipodium. 3)Formation of focal adhesions in the lamellipodium and adhesion to the substratum. 4)Stable contacts with the underlying surface prevent the membrane from retracting. There is a cytosolic flux forward. 5) The cell “ tail” eventually detaches and retracts into the cell body. Cell contraction as well as muscle contraction involve Actin and Myosin II 50 3- FORMATION OF THE CONTRACTILE RING AND CYTOKINESIS: cell in twois the daughter following Cytokinesis processcells that divides a mitosis. Each daughter cell must receive the same amount of cytoplasm and organelles. Contractile ring (Actin and Myosin II) Actin filaments A CONTRACTILE RING consisting of Actin filaments and Myosin II is assembled at the equator of the dividing cell. Its contraction pulls the plasma membrane progressively inward until it closes off and splits the cell in two. Cleavage furrow Myosin Daughter cells 51 What would happen if Myosin II is inhibited during mitosis ? When the myosin II is inhibited the cell is able to replicate its DNA but fails to divide. As a result the cell becames multinucleated. 52 STABLE MICROFILAMENT STRUCTURES  MICROVILLI :  Fingerlike extensions of the plasma membrane that are particularly abundant in cells involved in absorption such as the epithelial cells lining the intestine. The purpose of these thin protrusions is to increase the surface area available for absorption.  They do not possess intrinsic movement capacity  They are smaller than cilia (1/10 or 1/20 of the cilia size)  A single cell can have several thousand microvilli. ACTIN FILAMENTS with associated proteins MYOSIN I VILLIN FIMBRIN 53 STABLE MICROFILAMENT STRUCTURES  STEREOCILIA :  Similar to microvilli but longer  They are designed to detect and sense fluid motion (mechanosensors).  They tend to group in arrays or bundles.  Very important in auditory hair cells of the inner ear where they transform mechanical stimuli into electrical signals. 54 INTERMEDIATE FILAMENTS (IF) 55 INTERMEDIATE FILAMENTS  Intermediate size (10 nm)  Present in all animal cells  Highly stable polimers of fibrous proteins (there is NO polymerization and depolymerization).  They do not require ATP or GTP for assembly or disassembly.  They are composed of different proteins depending on the cell type : Epithelial cells : Keratins Neurones : neurofilament proteins Muscle cells : Desmin  Abundant in the cytosol of cells that are subject to mechanical stress FUNCTION: Intermediate Filaments DO NOT contribute to cell motility. Their functional role is to provide mechanical strength to the cell and to dissipate tensile forces to avoid cell or tissue damage. IF also provide support to the nuclear membrane. 56 DIFFERENT TYPES OF INTERMEDIATE FILAMENTS 57 INTERMEDIATE FILAMENT STRUCTURE All IF proteins have a central α-helical region flanked by two globular N- and C-terminal domains. The helical segments of two monomers interwind around each other to form a coiled-coil dimer with both N- termini on one side and both C- termini on the other side (PARALLEL DIMER). Two parallel dimers associate in an antiparallel orientation to form a staggered TETRAMER. Tetramers assemble end-to end to form PROTOFILAMENTS Protofilaments associate laterally to form PROTOFIBRILS Protofibrils wind around each other to form a ROPELIKE FILAMENT 58  Assembly of Intermediate Filaments does not require ATP or GTP.  Intermediate Filaments do not show structural polarity : both ends are identical.  The final structure can be easily bent but it is extremely hard to break : it is flexible but very resistant. 59 INTERMEDIATE FILAMENTS IN THE NUCLEAR LAMINA The NUCLEAR LAMINA is a fibrous meshwork that lines the inner face of the nuclear envelope. It is composed of Intermediate Filaments (made up of Lamins) and other associated proteins. LAMIN INTERMEDIATE FILAMENTS support and strengthen the nuclear envelope. 60 DYNAMICS OF NUCLEAR INTERMEDIATE FILAMENTS During the initial stages of mitosis the nuclear envelope breaks down and the nuclear lamina intermediate filaments disassemble in a tightly regulated process. At the end of mitosis the nuclear envelope forms again and the nuclear lamina re-assembles. 61 IF- BINDING PROTEINS : PLECTIN  Plectin can cross-link intermediate filaments to increase resistance.  Plectin can also bind intermedaite filaments to other cytoskeleton proteins and structures like microtubules and microfilaments Microtubules Intermediate Filaments Plectin 62 HUMAN DISORDERS ASSOCIATED WITH INTERMEDIATE FILAMENTS  Epidermolisis Bullosa Simplex : Caused by mutations in several Keratin genes. As a result, cells in the epidermis become fragile and easily damaged. In affected individuals skin is less resistant to friction and minor trauma and blisters easily. Normal Keratin Mutant Keratin  Amyotrophic Lateral Sclerosis (Lou Gehrig's syndrome) : It is a Motor Neuron Disease that has been linked to alterations in Neuron Intermediate Filaments and to accumulation and abnormal assembly of neurofilaments  Neurodegeneration  Laminopathies : Progeria, Muscular Distrophy 63 OVERVIEW OF CYTOSKELETON STRUCTURES 64 CELL JUNCTIONS TIGHT JUNCTIONS ADHERENS JUNCTIONS, DESMOSOMES FOCAL ADHESIONS HEMIDESMOSOMES GAP JUNCTIONS 65 INTRODUCTION  Physical interactions between cells are essential for the development and function of multicellular organisms.  Cells are held together by DIRECT cell-cell junctions or by INDIRECT cellextracellular matrix interactions.  Cell junctions are macromolecular structures that establish connections between the plasma membrane of adjacent cells or between the plasma membrane and the extracellular matrix.  Cell-cell interactions can be TRANSIENT ( such as those between cells of the immune system) or PERMANENT (such as those in epithelial sheets). The main functions of cell junctions are : Tight sealing and barrier formation Adhesion and Anchorage Cell-cell communication 66 FUNCTIONAL CLASSIFICATION OF ANIMAL CELL JUNCTIONS  TIGHT JUNCTIONS: They create a barrier that prevents the passage of fluids and molecules between cells or between different compartments.  ANCHORING JUNCTIONS: They are important in keeping cells together and providing structural cohesion in animal tissues. They include : Adherens Junctions Desmosomes Hemidesmosomes Focal Adhesions  GAP JUNCTIONS : They create a channel between neighboring cells that allows direct communication between them. 67 FUNCTIONAL CLASSIFICATION OF ANIMAL CELL JUNCTIONS 68 PROTEINS IMPLICATED IN ADHESION AND CELL JUNCTION : STABLE Claudine, occludine: tight junctions as Cell barriers Integrins. Dimers: (α and β chains). They form bonds with molecules of the extracellular matrix (focal adhesions and hemidesmosomes), but also with molecules of the immunoglobulin type. Cadherins. They are found as dimers on the surface of epithelial cells. forming bonds between contiguous cells. They participates in Adherent and desmosomal junctions. Connexins: Communicate the cytoplasm of adjacentcells NON STABLE. Selectins. They form links with carbohydrates in contiguous cells. They are involved in the movement of various types of blood cells that cross the endothelium to reach their destination. Immunoglobulins. (N-CAM, V-CAM, I-CAM) Links with other contiguous cell immunoglobulins, and with integrins. Nerve, endothelial and Inmune sistem-related cells. Neural crest loses N-CAM during migration. They hold cells together to form tissues α β 69 69 TIGHT JUNCTIONS  Specialized cell-cell adhesions with key functions in animal cells, particularly in epithelial cells  They form really tight associations between adjacent cells : Zonula occludens  They obliterate the extracellular space and help form a barrier that seals off body compartments such as the intestinal and stomach lumen or the bile duct in the liver. In other words, they prevent the leakage of molecules through the gaps left between adjacent cells.  Tight junctions are formed by a network of protein strands that extends around the entire circumference of the cell. Each strand is mainly composed of two transmembrane proteins, OCCLUDIN AND CLAUDIN that bind to similar proteins on the adjacent cell. These proteins also establish connections with actin filaments. 70 FUNCTIONS OF TIGHT JUNCTIONS in EPITHELIA 1.Maintain the polarity of epithelial cells by preventing the free diffusion of membrane proteins and lipids between the apical and the basolateral domains of the plasma membrane, and thus ensuring that these regions contain different membrane components. 2.Control the flow of solutes from the extracellular space by sealing the gap between cells. As a consequence highly selective and directional transport systems between apical and basolateral domains are required in epithelial cells. For example the transport of Glucose between the intestinal lumen and the blood supply. 71 In the Intestinal Epithelium extracellular fluids on both sides must be kept separate to maintain different concentrations of solutes and different fluid composition. Tight Junctions are absolutely essential for this. Extracellular Fluid Blood Cytosol Intestinal Lumen 72 ADHERENS JUNCTIONS (Cadherins bound to Actin Filaments) Anchoring interactions that maintain epithelial cells tightly bound. They are located close and basal to tight junctions and form a continuous adhesion belt around the cell called Zonula adherens. In these junctions E-cadherins establish cell-cell interactions through their extracellular domains and, at the same time, attach to intracellular actin filaments through their cytoplasmic domain. 73 DESMOSOMES : MACULA ADHERENS ( Cadherins bound to Intermediate Filaments ) They do not form a continuous belt but rather discrete spot-like structures. They can be located below the adherens junctions, but also on any other area of the lateral membrane. Two specialized cadherin proteins, DESMOGLEIN AND DESMOCOLLIN bind to each other (on different cells) through their extracellular domains and to some plaque-forming proteins (plakoglobin and plakophilins) through their cytosolic domains. In turn, these adapter proteins interact with intermediate filaments of the cellular cytoskeleton. 74 75 HEMIDESMOSOMES AND FOCAL ADHESIONS CELL-MATRIX JUNCTIONS  Hemidesmosomes : Discrete junctions between the basal membrane of epithelial cells and the basal lamina. They are mediated by INTEGRINS that attach to intermediate filaments through adaptor proteins.  Focal Adhesions : Discrete junctions betwen the plasma membrane of a cell and the extracellular matrix. They also involve INTEGRINS but in this case, this transmembrane proteins attach to actin filaments. 76 Proteins Involved in Anchoring Junctions Focal adhesions 77 GAP JUNCTIONS  Structures that provide a direct connection between the cytoplasm of adjacent cells and allow the free diffusion of ions and small water-soluble molecules between neighboring cells.  They are most abundant in non-epithelial cells but they can also be found in epithelia.  They are formed by transmembrane proteins called CONNEXINS, that combine to form a hexagonal hemichannel called CONNEXON consisting of 6 connexin molecules. When two connexons from adjacent cells align and join , a continuous aquous pore is formed.  GAP junctions couple both the metabolic activities and the electric responses of the cells they connect. 78 GAP junctions do not remain permanently open, they flip between open and closed states. Moreover, cells can regulate the permeability of their GAP junctions. For example, high intracelular Ca++ concentrations will close the channels. Extracellular signals can also regulate GAP junction permeability. 79 Gap junctions 80 Cell junctions found in a vertebrate epithelial cell 81 SUMMARY OF CELL-CELL JUNCTIONS FUNCTION TYPE MEMBRANE PROTEIN COMMUNICATION GAP JUNCTION Connexin SEALING TIGHT JUNCTION Claudin Occludin ANCHORAGE ADHERENS JUNCTIONS DESMOSOME Cadherin 82      SUMMARY Functions: anchor, mechanical support, cell shape, mov. organelles and cell Components: Filamentous structures formed by monomers Microtubules / alpha and beta tubulin (dimer, globular): from the centrosome or MTOC, pol / despol depending on GTP hidrolisis Microfilaments: actin (monomer, globular): connect and coat the inner plasma membrane and the nuclearouter membrane, pol / despol depending on ATP hidrolisis Intermediate Filaments; specific proteins (dimer.tetramer) stable, "reticular" appearance Functions: anchorage, mechanical support, cell shape, …. Componets: Monomers different types in different tissues Microtubules:  Structure: 13 protofilaments, of αβ tubulin. Pol / despol implies β tubulin = GTPase.  Stable structures of MT: cilia / flagella [9x2 + 2] and centrioles [9x3]. Centrosome: structure and composition (pericentriolar proteins , γTURC)  MT during mitosis: S-phase centrosome duplication  MT and vesicular transport: dynein and kinesin.  Molecules that affect pol / despol: colchicine, taxol, // catanin, catastrophin  Stabilizing MAPs [when MAPs are phosphorilated  separate from MT, depol] [dephosphorylated MAPs obound to MT, which stabilizes]  Associated pathologies: Tauopathies, Tau, a type of MAP, is hiperphosphorylated in Alzheimer's Microfilaments  Actin G (globular, soluble) and, F actin (polimerized in a microfilament)  Dynamic instability: Actin F, hydrolyzes ATP to ADP and the filament becomes unstable, it shortens end  Regulatory proteins: PROFILINE (+ assembly), THYMOSINE (-assembly) GELSOLINE, COFILINE (cut), CAPZ, TROPOMODULIN (stabilizers); FILAMIN FIMBRIN A-ACTININ (nets and bundles)  Role in cell movement, in the cytokinesis ring (together with myosin II), platelet coagulation, muscle contraction  Structural role in microvilli (together with vilin, myosin I) and sterocilia Intermediate Filaments  Support different forces, tensions, ≠ types depending on the cell, neurofilaments, keratin, Some like the lamina, are in all  Dimertetramerprotofilamentprotofibrils Rope like IF  PLECTINS: binding to other elements of the cytoskeleton  Associated pathologies: ALS, epidermolysis bullosa, laminopathies 83 Cell junctions  Cell-C and matrix cell junctions: types, location, molecules involved, biological role - Non stable (Ig, selectins) - Stable c-c: CommunicatIion GJIC, occludens Z ocludens. Ex. Polarity of epithelial c Claudin, occludin, ZO 1,2,3 Anchor. Z adherens (adhesion and desmosome)  cadherins, desmogleins desmoplakins actin - Stable C-matrix : focal adhesions and hemidesmosomes integrins, intermediate filaments 84