Medical Biology Cell Fractionation Methods PDF

MED100-I MEDICAL BIOLOGY Methods for examination of cells II-Fractionation of cells and analyzing their molecules Assoc. Prof. Özlem KURNAZ GÖMLEKSİZ  II-Fractionation of cells and analyzing their molecules cells are disrupted and their organelles and macromolecul...

MED100-I MEDICAL BIOLOGY Methods for examination of cells II-Fractionation of cells and analyzing their molecules Assoc. Prof. Özlem KURNAZ GÖMLEKSİZ  II-Fractionation of cells and analyzing their molecules cells are disrupted and their organelles and macromolecules isolated in pure form II-Fractionation of cells and analyzing their molecules to study certain organelles from a cell (the mitochondria , the ) Isolating these organelles involves a variety of procedures collectively called cell fractionation. As a method for studying processes within organelles, cell fractionation has both advantages and disadvantages. cells are disrupted and their organelles and macromolecules isolated in pure form  Cells can be disrupted in various ways osmotic shock or ultrasonic vibration forced through a small orifice, or ground up break many of the membranes of the cell If carefully applied, however, the disruption procedures leave organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes largely intact. The suspension of cells➔ homogenateor extract  Cell fractionation is where a cell are broken up and its components and organelles are separated so that scientist can observe them in isolated form. the separation of homogeneous sets, usually organelles, from a larger population of cells. Cell fractionation methods Involve the homogenization or destruction of cell boundaries by different mechanical or chemical procedures, followed by the separation of the subcellular fractions according to mass, surface, and specific gravity Steps of subcellular fractionation 1. Homogenization 2. Differential centrifugation 3. Further separation and purification by density gradient centrifugation 4. Collection of fractions 5. Analysis of fractions Homogenization or Cell Disruption Chemical : alkali, organic solvents, detergents Enzymatic : lysozyme , chitinase Physical : osmotic shock, freeze/thaw Mechanical : sonication , homogenization, French press Centrifuges rotate at high speed  Centrifugation is the first step in most fractionations, but it separates only components that differ greatly in size. A finer degree of separation can be achieved by layering the homogenate as an arrow band on top of a dilute salt solution that fills a centrifuge tube. When centrifuged, the various components in the mixture move as a series of distinct bands through the salt solution, each at a different rate, in a process called velocity sedimentation.  For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles) finds itself on top of a lighter one (the salt solution) This is achieved by filling the centrifuge tube with a shallow gradient of sucrose prepared by a special mixing device; the resulting density gradient.  by using an instrument known as the ultracentrifuge. In which extracts of broken cells are rotated at high speeds  When a centrifugal force is applied to an aqueous mixture, components of larger size and density will sediment faster Low speed centrifugation is used to separate intact cells from medium High speed centrifugation can be used to separate subcellular components Isolation of components of living cells by differential centrifugation In 1934 First isolation of Mitochondria (from liver) by Bensley 1937 → First Chemical analysis of Mitochondria has been made  Cells can be broken up in various ways: (osmotic shock or Ultrasonic vibration or ground up in a blender,homogenizer) The organ or tissue is cut into small fragments They then immersed in an appropriate solution placed in a Homogenizer (A glass cylinder within which a turning rod) Cells are mechanically disrupted by grinding  Homogenization breaks the cell membranes Liberates the organelles and other cellular structures into the solution then Cell suspension is centrifuged at high speed Heaviest particles are sedimented first particles of lower densities can be sedimented when we increase the speed progressively. Organelles and Macromolecules Can Be Separated by Ultracentrifugation Made it possible to isolate pure fractions of Nucleus Nucleolus Mitochondria Lysosomes Microsomes ribosomes We have obtained our knowledge on molecular composition of cell components  Method of Differential Centrifugation: 1. Cut tissue in an ice cold isotonic buffer. It is cold to stop enzyme reactions, isotonic to stop osmosis and a buffer to stop pH changes. 2. Grind tissue in a blender to break open cells. 3. Filter to remove insoluble tissue 4. Centrifuge filtrate at low speeds ( 1000 X g for 10mins)→ This pellets the nuclei as this is the densest organelle 5. Centrifuge at medium speeds ( 10 000 x g for 30 mins )→ This pellets mitchondria which are the second densest organelle 6. Centrifuge at high speeds ( 100 000 x g for 30 mins)→ This pellets ER, golgi apparatus and other membrane fragments 7. Centrifuge at very high speeds ( 300 000 x g for 3hrs) This pellets ribosomes  CELL-FREE SYSTEMS ( Fractionated cell extracts that maintain a biological function) Molecular mechanisms involved in cellular processes can be studied In 1949 Isolated myofibrils from skeletal muscle cells contract upon the addition of ATP In 1955 Ciliary beating In 1954 First cell free-system to carry out protein synthesis (in vitro translation) CELL-FREE SYSTEMS In 1983 In vitro cell cycle (extracts from frog eggs) In 1984 Golgi vesicle trafficking in vitro using a cell free system to analyse the molecular details of DNA replication DNA transcription transport along microtubules Much of what we know about the molecular biology of the cell has been discovered by studying cell free systems.  MED100-I MEDICAL BIOLOGY Assoc. Prof. Özlem KURNAZ GÖMLEKSİZ Dept. of Medical Biology E-mail: [email protected]  Reference Books 1. The Cell – A Molecular Approach Geoffrey M. Cooper 2. Molecular Biology of the Cell B. Alberts, D Bray, J. Lewis, M. Raff, Keith Roberts, JD. Watson 3. Molecular Cell Biology J. Darrnell, H. Lodish, D Baltimore, Thomas D Pollard, WC Earnshaw 4. Medical Cell Biology Steven R. Goodman 5. Molecular and Cellular Biology Stephen L Wolfe 6. Human Molecular Biology RJ Epstein Introduction to Cell Biology What is a cell CELLS: are basic morphological and functional units of the body (They are basic building blocks of all living organisms) THE CELL Mammalian tissue is made up of Cells Intercellular or extracellular substances (They lie between cells to support and nourish them. i.e. collagen, reticular fiber, elastin, ground substance) Tissue fluid (located between and around cells) CELLS: are basic morphological and functional units of the body The Cell Cells are too small invisible to the naked eye Development of cell biology had to await the development of a magnifying device: Light microscope Cell Biology begins with the Light microscope. First microscopic observations have been made during 17th century The term Cellula first used by (Robert Hook) 1665 when he first observed a piece of cork under a microscope Which is made up of small chamber like structures ,cellula General meaning of the term cell (cellula): Small room, a hut 1674 first microscopic observations have been made by Leeuwenhoek He observed protozoa,bacteria and sperm Pictures of rabbit,dog,bull,cock sperms drawn by Leuvenhoek  PROTOPLASM: The living substance of the plants and animals (J.Purkinje→1840) (Total living matter: nuclear region + cytoplasm) The smallest unit of protoplasm which is capable of independent existence is the cell. 1833 R.Brown → introduced the term nucleus CELL THEORY ( Schleiden 1838 and Schwann 1839) ▪ Cells are the fundamental units of both structure and function in all living organisms. ▪ All living organisms are composed of cells and their products. ▪ Cells arise only from preexisting cells. Cells are small The size of a cell varies. The biggest human cell, the egg, (150-200µm) Smallest cell :granular cells of cerebellum 4-5µm Size of a typical animal cell = 20 - 30 µm  How many cells are there in the human body How many cells are there in the human body Human body is composed of trillions of cells METHODS FOR EXAMINATION OF CELLS  How cells are studied I- Examination of the cell as a whole (without disruption) A- Examination of living cells (Fresh tissues) B- Examination of killed and preserved tissues and cells II-Fractionation of cells and analyzing their molecules I-Methods for examination of living cells (Vital examination) Cells are small and complex Size of a typical animal cell = 20 - 30 µm Looking at the structure of cells in the microscope, Direct observation Examination of living cells under a microscope is the oldest one of the techniques Development of the first Light Microscope Cell biology began with the light microscope A-Examination of living cells (Fresh tissues) Cell and Tissue Cultures Isolating cells and growing them in culture (Cell Culture) Living cells → can be suspended in an appropiate liquid (i.e.saline solution) Examined under the L.M ↓ They will soon die Prolonged study of living cells can be made ↓ by culturing them in solutions (containing necessary nutrients) to keep them alive  First tissue culture (1907) by Ross Harrison Isolated fragments of spinal cord of a frog Kept the tissue alive in lymphatic fluid (first culture media) Demonstrated that nerve fibers could grown out ( nerve cells extended long thin processes) Classical Culture media: Blood serum (blood clot) Embryo juice (growth stimulant) (an extract of embryonic tissues) Fragments from spinal cord + Culture media 1day Axons growing in culture! In Primary Cultures Cells and tissue fragments are isolated and maintained in a culture media (In vitro experiment) Cells attach to the surface of the culture dish Migrate radially from the explanted tissue Proliferate in a few days Several times the diameter of the original tissue  Cell cultures have played an important role in the development of cell biology and molecular cell biology Animal cells are more difficult to culture than microorganisms they require more nutrients But various types of cells can be cultured succesfully  Mammalian cell culture medium Rich media are required (contain necessary nutrients) ▪ Amino acids (10 amino acids called essential amino asids can not be synthesized by vertebrates and must be obtained from diet; Arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine) must be supplied ▪ Vitamins ▪ Various Salts ▪ Glucose ▪ Serum (contains various factors needed for proliferation of cells; growth factors)  Mammalian cell culture medium ◼ Most animal cells can only grow on special solid surfaces (adhere to and grow on glass and treated plastics ◼ Medium should be changed frequently ◼ Aseptic technique is necessary to avoid contamination (Antibiotics) ◼ We use a cell culture hood. laminar-flow hood ◼ Temperature (37 ºC) ◼ (Cultivation takes place in a CO2 incubator, in which not only the temperature, but also the humidity and Gases (O2, CO2 ) content, must be controllable) ◼ pH 7,4 (phenol red: indicator dye) Laminar flow cabinet clean bench Primary neuron culture from brain cortex Brain research Lab in Cerrahpasa Medical faculty Culture Conditions A medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals) Growth factors Hormones Gases (O2, CO2) A regulated physico-chemical environment (pH, osmotic pressure, temperature) Most cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate  Given appropriate surroundings, ( in an environment that is as natural as possible.) Most animal and plant cells can live, multiply and express differentiated properties in a culture dish The cells can be watched continuously under the microscope and the effects of adding or removing specific molecules i.e hormones or growth factors can be explored A primary cell culture (is derived from normal animal cells) A primary cell culture have a limited life span Divide ~ 25-50 times (will not continue to grow indefinitely) Stop dividing (a process called “cell senescence” “and die Cell lines Eukaryotic cell lines are a widely used cell source for experiments Transformed cells (including tumor cells) Grow indefinitely in culture (immortal cells) A culture derived from single transformed cell is called “ cell line” (transformation is a change that causes them to behave as tumor cells)  Phase-contrast micrograph of cultured cells Cells in culture. (A) Phase-contrast micrograph of fibroblasts in culture. (B) Micrograph of myoblasts in culture shows cells fusing to form multinucleate muscle cells. (C) Oligodendrocyte precursor cells in culture. (D) Tobacco cells Microdissection techniques allow selected cells to be isolated from tissue slices  Applications of the cell culture 1. Embryonic organs can be cultured (a small embryonic bone can grow in length and undergo calcification outside of the body) Morphogenesis 2. The study of cancer cells. (Malignant transformation) 3. Cell to virus relations 4. Cytogenetic research (the study of human chromosomes ) (Diagnosis of diseases caused by abnormalities of chromosomes) 5. Cell-cell interactions 6.Cell nutrition importance of enviromental effects on the tissue differentiation.  Embriyonic stem cells Embriyonic stem (ES) cells are most promising cell lines –from medical point of view- These cells removed from inner cell mass of the early mouse embryo can proliferate indefinitely these cells can give rise to all the cell types in the body. ES cells can be derived from early human embryos these cells might be used to replace and repair damaged mature human tissues. It may be possible in the future to use ES cells to produce specialised cells for therapy To replace skeletal muscle fibers that degenerate in muscular dystrophy patients Nerve cell that die in patients with Parkinson’s disease Insulin secreting cells that are destroyed in type I diabetics  Escherichia coli is the best understood cell in the world of biology Most popular bacterium Saccharomyces cerevisiae (yeast) most frequently used single cell eukaryote (most primitive eukaryotic cell) Studies with bacteria and yeast established the basic principles of molecular biology  The use of hybrid cells (Hybrid cells are produced by Fusion of animal cells) What is cell-cell fusion Cell fusion: membrane merging and cytoplasmic mixing of two cell types i.e. Human cell and Mouse cell Is cell fusion a normal process? We need specific substances that promote cell to cell fusion A viral glycoprotein promotes cell fusion (Fusion proteins) Polyethylene glycol is also used  Applications of cell fusion Mouse cell + human cell →Fusion (Hybrid cell) Heterokaryon Nuclei also fuse (Chromosomes comes together from both cell) Human and mouse hybrids grow and divide Gradually loose human chromosomes in random order The production of hybrid cells membrane merging and cytoplasmic mixing Nuclei also fuse chromosomes come together  Hybrid cells grow and divide.Gradually loose human chromosomes In Somatic hybrids, human cell can be fused with mouse (some other organism) cells. At first they form heterocaryons (cells with two or more nuclei) but ,they form hybrid cells (each with one fused nucleus). The treatment of cells with polyethylene glycol or inactivated Sendai virus is what causes the fusion to occur. In human-rodent hybrid cells there is a progressive loss of the human chromosomes. This feature of hybrid cells allows researchers to isolate a single human chromosome, or even a part of a chromosome.  Applications of cell fusion 1. Genetic analysis: A cell line containing a single human chromosome (i.e Chromosome 11 –produces human- insulin ) Result: Insulin gene is located on Chromosome 11 (first gene mapping) Used to map genes to specific chromosomes  Applications of cell fusion 2. Production of Monoclonal antibodies Antibodies for use as tools in cell biology and clinical medicine) To detect specific proteins by immunocytochemistry, ELİSA, RIA methods B lymphcytes produce antibodies A clone of cells from a single antibody secreting B lymphocyte can be obtained, These cells will produce this specific antibodies antibodies can be isolated from the culture medium  Hybridoma cell lines are factories that produce monoclonal antibodies B lymphcytes produce antibodies Problem; B lymphocytes have limited life span in culture To overcome this problem B lymphocytes from an immunized mouse are fused with cells derived from an immortal B lymphocyte tumor Hybrid cells have both the ability to make a specific antibody and to divide indefinitely (immortal) These hybridomas provide a permanent and stable source of a single type of monoclonal antibody  Preparation of hybridomas that secrete monoclonal antibodies against a particular antigen Mechanical Micromanipulation (Application of Microsurgical procedures to the living cells) (Microsurgical procedures are applied to the living cells) Technical problems: Cells are so small they must be magnified. ( x 100-1000) Cells must be maintained in a physiological environment. Which cells are useful for this method ? The cells in the culture (spread on glass surface) We need extremely minute instruments. (microneedles, micropipettes, microinjectors) We need an operation chamber. Microinstruments are localized within an operation chamber  Studies: On the elasticity and viscosity of protoplasm The dependence of cell function upon the nucleus. To demonstrate that materials can pass from the nucleus into the cytoplasm. To inject minute amounts of substances directly into the cytoplasm of fertilized mammalian eggs. (Volumes a small as 1 picoliter)1 picoliter : 1µµl:1/50 milion of a drop The effect of these drugs upon development can be observed.  In vitro fertilization In vitro fertilization (test tube baby) (IVF) is a process by which egg cells are fertilized by sperm outside the body,(in vitro) Direct microinjection of DNA into cell nucleus Transgenic mice produced by microinjection of cloned DNA into the pronucleus of a fertilized egg.  MED100-I MEDICAL BIOLOGY SPECIALIZATIONS OF THE CELL SURFACE (CELL SURFACE MODIFICATIONS) Assoc. Prof. Özlem KURNAZ GÖMLEKSİZ Dept. of Medical Biology E-mail: [email protected]  Epithelial cells show polar differentiation They have apical, lateral and basal sufaces They exibit modifications on their surfaces  Apical=luminal=Free surface Epithelial cells show polar differentiation Lateral surface Lamina propria Basal surface (connective tissue ) APICAL SURFACE MODIFICATIONS The surface of the most cells have extensions They are used in cell movement, absorbtion. I- SPECIALIZATIONS OF FREE SURFACE (APICAL SURFACE ) 1-Microvilli 2-Cilia, flagella 3-Stereocilia  1-Microvilli Finger-like projections from the cell surface Function: increase the surface area for absorption Localisations (cells specialized for absorbtion ) 1-Intestinal epithelium (Striated border ) (LM) 2-Proximal tubule of the kidney (Brush border) (LM) 3- Epithelium of gall bladder  Microvilli *Finger-like protrusions 1  in length 0,1 diameter (Can be seen with L.M) *Enclosed in an extension of the plasma membrane *Contains a bundle of straight parallel filaments (20-30 Actin filaments ) *Actin filaments extend 0,5 down into the apical cytoplasm and enter into terminal web Light microscopic appearience of microvilli (a border of vertical striations) A microvillus. (A) Contains A bundle of parallel actin filaments that extend 0.5 down into the apical cytoplasm and enter into terminal web. Actin filaments cross-linked by the actin- bundling proteins; villin and fimbrin, forms the core of a microvillus Microvillus Actin filaments are cross linked into closely packed bundles by actin bundling proteins; fimbrin and villin Actin filaments are attached to the plasma membrane by lateral arms consisting of myosin I and calmodulin Glycocalix is thicker around the microvilli Striated border Glycocalix PAS +  2-Cilia and Flagella Cilia are eyelash or hair like processes from the cell surface (can be seen in LM) -Motile processes Longer than microvilli (5-10 long, 0.2 in diameter) Under the electron microscope they have a complex internal structure (composed of microtubules) 250 or more cilia (in each cell) arranged in parallel rows Function; to move the fluid over the surface of the cell Cilia (L.M) Scanning electron microscope (SEM)  Where are they found? Localisations: Uterine tubes Respiratory tract (oviduct) 1-Surface of epithelial cells of the upper respiratory tract 2- Epithelial cells of the uterine tubes (oviducts ) 3- Epithelial cells of the efferent ducts (Ductus efferentes) The efferent ducts connect the testis with the epididymis.  Cilia * Hair –like protrusions from the cell surface At the base of each cilium a dense granule (Basal body) is seen With E.M  Ciliary movement (rapid back and forth movement) is constant in direction In living cells Cilia beat in a rhythmical wave –like manner (can be observed with phase-contrast microscope) function;to move the fluid over the surface of the cell Protozoa use cilia for locomotion Functions in our body: 1-to move mucus over epithelial surfaces in respiratory epithelium toward the mouth. (Clear the mucus together with dust particles, dead cells and bacteria from respiratory passages) 2- In uterine tubes to transport the ovum toward the uterus. 3- In ductus efferentes (efferent ducts) to propel the spermatozoa toward the epididymis Respiratory epithelium. The regular, coordinated beating of the cilia moves the mucus over the surface of the cells out of the airways, mucus carries dust particles, bacteria that is stuck to it. Movement of Cilia …………. Backword movement Cilia Cilia have a complex internal structure A characteristic arrangement of microtubules called “ Axoneme’’ 9+2 microtubule complex 9 peripherally located double microtubules (doublet) and 2 centrally located single microtubules (singlet): ( 9+2 ) The arrangement of microtubules in a cilium or flagella A) Electron micrograph of a cilium shown in cross section, illustrating the “9 + 2” arrangement of microtubules (Axoneme) (B) Diagram of the parts of a flagellum or cilium. In each doublet ; Microtubule A is complete, it consist of 13 protofilaments in its wall (it has «O» shaped cross section) Microtubule B is incomplete, ıt consist of 10-11 protofilament, it has a C shaped cross section. It is fused to Microtubule A and closes the defect in its Wall.  Dynein arms are arranged along the length of the microtubule.  They are formed by a protein called “ dynein’’ and contain ATPase (ATP splitting enzyme) activity. “Nexin links’’ Nexin links attach each microtubule A to the microtubule B of the adjacent doublet.  Nexin links are composed of an elastic material called “nexin’’  Responsible for recovery stroke (backward movement) (In longitudinal section) Each cilium is covered by an extention of plasma membrane 1-Tapering tip 2-Cylindrical shaft 3-Basal body (located in the apical cytoplasm) MECHANISM OF CILIARY MOVEMENT Cilia beat in a rhythmical wave-like manner’’ Old concept: Ciliary movement is based on the contraction of microtubules (Microtubules are capable of shortening ) IT WAS WRONG!!! Sliding microtubule mechanism  Sliding microtubule mechanism Satır, studied the mechanism of ciliary movement (1968) He examined cross sections near the tips of cilia in different phases of beating Doublets of the bend cilium terminate at different levels Result: ciliary movement is based on sliding microtubule mechanism (sliding of douplet microtubules relative to one another. He examined cross sections near the tips of cilia in different phases of beating  Sliding Microtubule Mechanism (Satır) *If a cilium is straight doublets terminate at the same level *If a cilium is bent toward doublets 5 and 6 5 and 6 project farthest Doublet 1 terminates first If movements were produced by microtubule shortening, microtubules on the concave side (5 and 6) Should be shorter. Result: ciliary movement is based on sliding microtubule mechanism (sliding of douplet microtubules relative to one another. Ciliary bending occured without shortening but sliding of douplet microtubules  Result: Ciliary and Flagellar Beating Are Produced by Sliding of Outer Doublet Microtubules relative to one another. Sliding Microtubule Mechanism (Satır ) To day accepted mechanism: A cilium bends along the axis by a type of ” Sliding Microtubule Mechanism’’ between microtubules It is similar to that seen between myofilaments in striated muscle fibers  Cilia and flagella, from which the plasma membrane has been removed can beat when ATP is added; this in vitro movement is the same as observed in living cells. (Cell free system) The active sliding occurs all along the axoneme, Dynein is essential for motility of cilia and flagella Afzelius 1978 Depending on Clinical and Electron Microscopic observations on a congenital form of human infertility Afzelius showed that Dynein is essential for motility of cilia and flagella  Kartagener’s syndrome; Chacterized by 1- Respiratory tract disorders Bronchiectasis (chronic dilation of bronchi ) 2-Chronic sinusitis 3-Situs inversus totalis (complete transposition (right to left reversal) of the thoracic and abdominal organs.(Heart being in right,liver being in left 4-Male infertility Normal number of spermatozoa but no motility Afzelius 1978 examined the sperm of a patient with Kartagener’s syndrome with Electron Microscope  Afzelius’s Experiment 1978 Electron Microscopic observation of immotile sperm flagellum of patients with Kartagener’s syndrome: Showed the absence of dynein arms EM observation of bronchial biopsies showed no dynein arms in ciliary axonemes Result: Dynein is essential for motility of cilia and flagella Kartagener syndrome is a genetic disease There is a congenital defect in the synthesis of dynein  Males with Kartagener’s syndrome are sterile Sperm was immotile Dynein is absent from axoneme of sperm (EM) Immotility is due to the absence of Dynein arms He observed cilia from respiratory tract Cilia was also immotile (same defect;cilia do not have dynein) there is no transport of mucus in the tracheobranchial system Patients suffer from respiratory tract disorders Dynein –Walking Model *Dynein appears to “ walk’’ along the adjacent doublet Experiment: *Proteolytic enzymes digest nexin links and radial links *The addition of ATP produces a sliding movement of the dynein bridge along the B tubule of the adjacent doublet *Doublets move relative to each other by the motor activity of axonemal dynein *Radial linkers convert the sliding of microtubules into bending of cilia The bending of an axoneme. (A) When axonemes are exposed to the proteolytic enzyme trypsin, the linkages holding neighboring doublet microtubules together are broken. In this case, the addition of ATP allows the motor action of the dynein heads to slide one pair of doublet microtubules against the other pair. (B) In an intact axoneme (such as in a sperm), sliding of the doublet microtubules is prevented by flexible protein links. The motor action therefore causes a bending motion, creating waves or beating motions, Sliding of peripheral dublets after proteolytic digestion of nexin links. DYNEIN PRODUCES MICROTUBULE SLIDING Aafter proteolysis In the presence of of nexin links nexin links sliding is converted to bending  DYNEİN First identified microtubule motor, first isolated in 1965 Extremely large protein *Dynein arms form temporary cross bridges between microıtubule A of one doublet and microtubule B of the adjacent doublet *During sliding they undergo a cyclic break and reattachment *Formation of cross bridges is ATP dependent Ciliary dynein. (Ciliary (axonemal) dynein is a large protein assembly composed of 9–12 polypeptide chains B) Freeze-etch electron micrograph of a cilium showing the dynein arms projecting at regular intervals from the doublet microtubules; only the A microtubule is shown. (B, courtesy of John Heuser.) Ciliary movement Requires 1-ATP 2-Ca ions , Mg ions  Basal Body At the base of each cilium a dense granule (Basal body) They are Cylindrical structures of 0.2 m wide and 0.4 m long Nine sets of triplet microtubules, form the wall of the basal body Basal body (LM) Basal bodies. (A) Electron micrograph of a cross section through three basal bodies i (B) Each basal body forms the lower portion of a ciliary axoneme and is composed of nine sets of triplet microtubules, each triplet containing one complete microtubule (the A microtubule) fused to two incomplete microtubules (the B and C microtubules). Other proteins (shown in red in B) form links that hold the cylindrical array of microtubules together.  Basal body At the base of a cilium central pair of single microtubules terminates Each of peripheral doublets is contunious with a triplet microtubule of basal body Three microtubules are fused together and form a triplet microtubule Basal body resembles a centriole but it contains some accessory structures such as basal foot and rootlet Rootlet;Strands of fibrous material extend from basl body into the apical cytoplasm Basal foot:Another fibrous material extends laterally Basal foot rootlet Cilia Longitudinal section Basal body=Centriole+ accessory structures (basal foot +rootlet)  Each triplet containing one complete microtubule (the A microtubule) fused to two incomplete microtubules (the B and C microtubules).  Basal body play an important role in organization of the axoneme microtubules. They have nine triplets of microtubules. Adjacent triplets are linked at intervals along their length Cilia and flagella grow from basal bodies that are closely related to centrioles  Ciliary movement is important during development.  Dynein arms are absent from cilia in patients with Kartagener’sSyndrome  Nodal Cilia can not move during development and internal organs can not be located at their normal positions  Helical beating of cilia at the node, and the origins of left-right asymmetry.  (A) The beating of the cilia drives a current towards one side of the node Various signal proteins are produced in this neighborhood, and the current is thought to sweep them toward one side,  (B) The asymmetric expression pattern of one such signal protein—called Nodal—in the neighborhood of the node (lower two blue spots) in a mouse embryo at 8 days of gestation,  FLAGELLA Flagellum propels sperm 1- Longer than cilia (100-200 μ ) Longest flagella are those of mammalian sperm 2- Same internal structure with cilia (Axoneme 9+2 ) 3-Different type of movement (undulating wave type of movement) 4- Less in number (one or two in a single cell) 5- Mammalian spermium contains 9 additional dense fibers arround the axoneme (9+9+2) (protective function) The contrasting motions of flagella and cilia. (A) The wave-like motion of the flagellum of a sperm cell (B) The beat of a cilium, Flagellum of mammalian spermium (cross secion) Scanning electron micrograph of a human sperm contacting a hamster egg.  STRUCTURE OF MICROTUBULE Microtubules are hollow tube like or pipe like structures Wall of the microtubule composed of 13 protofilaments Proto filaments are composed of Tubulin subunits (dimer) Dimer→ Tubulin α and Tubulin beta MICROTUBULES  interphase sentrosome ciliated cell Cilia/flagella Basal body Dividing cell spindle Nerve cell sentrosome axon  Centrioles. (A) Electron micrograph of an S-phase mammalian cell in culture, showing a duplicated centrosome. Stereocilia 1. Long and irregular microvilli 8μ (EM) 2. Have no microtubule containing internal structure 3. Contain poorly developed microfilaments 4. No motility 5. No basal body Function: Increase the cell surface for absorbtion Localisation: Ductus epididymis, ductus deferens MED101 MEDICAL BIOLOGY Cell-Cell Junctions Cell- Matrix Junctions Cell adhesion molecules Assist. Prof. Özlem KURNAZ GÖMLEKSİZ E-mail: [email protected] Lateral Surface Modifications Cell-Cell Junctions  The cells of most tissues are bound to one another by cell-cell junctions They are abundant in epithelial tissues. (more than 60% of the cell types in the vertebrate body are epithelial) Cell-cell junctions are found on lateral surface of epithelial cells; lateral surface modifications) Epithelial tissues cover the surfaces and line the cavities. Form protective layers) Epithelial cells show Apical=luminal=Free surface polar differentiation Junctional complex Lateral surface Lamina propria Basal surface (connective tissue ) Functions of cell-cell Junctions 1-cell to cell attachment (adhesion) 2-to form barriers that prevent the free passage of substances and cells from lumen to the blood circulation. 3-Cell to cell communication  CELL- CELL JUNCTIONS Cell junctions can be classified into three functional groups: I-Occluding junctions (tight junctions)(Barrier) II-Anchoring junctions (Adhering Junctions)(Cell-cell Attachment) Z.adherens and Desmosome III-Channel-forming junctions (Communicating junctions) Gap Junction CELL- CELL JUNCTIONS Cell to cell junctions between the epithelial cells  Microvillus Tight junction Adherens junctions Desmosomes Gap junction Hemidesmosome Basal Lamina Cell-cell and cell-matrix junctions  I-Tight junction=Zonula occludens Occluding junction Located just below the apical surface. Functions; 1-Seals neigbouring cells together prevents passage of molecules (including ions) from lumen to the blood circulation forms a barrier 2-Separates the apical and basolateral domains of the plasma membrane prevents the free diffusion of membrane components (Lipids and proteins)(restrict lateral movement of membrane proteins) Tight junctions:(Zonula occludens or Occluding junction) Tight junctions are the closest contacts between adjacent cells. Tight junctions prevent diffusion of membrane proteins and glycolipids between the apical and basolateral regions of the plasma membrane E.M micrograph of junctional complex Tight junctions are the closest contacts between adjacent cells. It was originally described that outer leaflets of the plasma membranes of the adjacent cells are fused To day it is clear that membranes do not fuse membranes of adjacent cells come together at periodic intervals (focal connections) ,  Tight junctions are located just below the apical surface. Continuous around the entire periphery of the cell (zonula) There is a very narrow intercellular space at the level of tight junctions EM shows that membranes of adjacent cells come together at periodic intervals (focal connections) , but membranes do not fuse. A-Schematic drawing shows a tight junction might be formed by the linkage of rows of protein particles. tight junctions consist of anastomosing network of protein strands higher magnifications demonstrate that tight junction strands consist of transmembrane proteins B-Freeze-fracture preparation of tight junction zone between two intestinal epithelial cells. C-The EM micrograph of a tight junction. Freeze-fracture images revealed that tight junctions consist of anastomosing network of strands At higher magnifications tight junction strands consist of transmembrane proteins (rows of transmembrane proteins): of claudin, occludin) Tight junctions are usually located just below the apical microvillar surface. A.Electron micrograph of a thin section of endothelial cells. B.Electron micrograph of a replica of a freeze-fractured cell. C.İnterpretive drawing showing the strands at points of contact as rows of transmembrane proteins. Tight junctions restrict the diffusion of all solutes larger than about 1.8 nm in diameter. Experimental demonstration that tight junctions prevent passage of water-soluble substances.  Both of these proteins bind to similar protein on the adjacent cell thereby sealing the intercellular space Cytosolic tails of Claudins and occludins associate with intracellular proteins of zonula occludens (ZO) which link the tight junction to the actin cytoskeleton. Tight junctions are responsible for Blood-Brain barrier and Blood –Testes barrier  The blood- brain barrier Tight junctions are responsible for Blood-Brain barrier and Blood –Testes barrier Endothelial cells of capillaries in the brain and spinal cord are joined by continuous tight junctions. Thus,form a physical barrier, called the blood-brain barrier, separates the blood from the nervous tissue. Prevents passage of substances from blood to the brain Helps to protect the brain, but it also creates difficulties in treating brain disorders Schematic diagram of the blood- brain barrier Endothelial cells of capillaries in most regions of the brain and spinal cord are joined by continuous tight junctions.Thus,form a physical barrier,called the blood-brain barrier, separates the blood from the nervous tissue. Inflammatory states such as meningitis may reduce the integrity of the blood-brain barrier.  Claudin mutations claudin 16 mutations cause a rare human renal magnesium wasting syndrome characterized by hypomagnesemia.  impaired reabsorption of Mg and Ca in the thick ascending limb of Henle's loop. excessive urinary loss of magnesium and calcium and progressive renal failure.  Mutations in claudin-16 and 19 can both cause this syndrome  A mutation in the claudin 14 gene causes hereditary deafness.  The paracellular pathways in the inner ear and in the kidney are predominant routes for transepithelial cation transport.  Mutations in claudin-14 cause nonsyndromic recessive deafness 2-Anchoring junctions (Adhering junctions)  Anchoring junctions (Adhering junctions) Functions: Cell to Cell adhesion Binding of cytoskeleton to the cell surface  Anchoring Junctions Composed of two classes of proteins. 1-Proteins providing cell-cell adhesion (Transmembrane adhesion proteins) CADHERİNS; Cell to Cell Adhesion 2-Intracellular anchor proteins (vinculin, alpha-actinin, catenin, desmoplakin) bind the cytoskeleton to the junctional complex The construction of an anchoring junction from two classes of proteins.  II- Anchoring junctions (adherens junctions There are two different forms: 1.ADHERENS JUNCTIONS (Zonula adherens) (are anchorage sites for actin filaments) and 2-DESMOSOMES (macula adherens) are anchorage sites for intermediate filaments) both hold cells together and are formed by transmembrane adhesion proteins that belong to the cadherin family. I- ADHERENS JUNCTION, Zonula adherens) Surrounds the entire periphery of epithelial cells near their apical surface. Just below the Z. Occludens (Belt –like ) (Zonula) Adhesion belts link neighbouring cells together Maintains the physical integrity of the epithelium. In Heart muscle similar junction called Fascia adherens which connect cardiomyocytes to one another Adherens junction = Zonula adherens. Electron micrograph from the intestinal epithelium. α and ß-catenin link the cytoplasmic domain of E-Cadherin to actin filaments. A contractile bundle of actin filaments running along the cytoplasmic surface of the junction Actin filament bundles are attached by intracellular anchor proteins to cadherins, Cadherins are transmembrane proteins and their extracellular domains bind to those of the cadherins on the adjacent cell In this way the actin filament bundles of adjacent cells are tied together Adherens junctions in the form of adhesion belts, between epithelial cells in the small intestine.  1. Cell membrane 2. Cell membrane α-catenin CADHERIN p120 β-catenin Vinculin α-Actinin Actin filament Outside of cell Adherens junction  Role of adherens junctions in early development The controlled contraction of the bundles of actin filaments running along the adhesion belt cause epithelial cells narrow at their apical domain and helps the epithelial sheet invaginate and form a tube (neural tube) in early vertebrate development The folding of an epithelial sheet to form an neural tube Functions They provide strong adhesion between adjacent cells They form attachment sites for actin filaments of the cytoskeleton to the cell surface Important role in vertebrate development  II-Desmosomes (macula adherens)  Desmosomes are buttonlike points of tight adhesion anchorage sites for intermediate filaments) Desmosomes are the strongest points of cell adhesion that provide mechanical binding Desmosomes and hemidesmosomes A desmosome between epithelial cells. Cadherin proteins (desmoglein and desmocollin) bind via anchor proteins (desmoplakin,plakoglobin,plakophilin to intermediate filaments. Desmosomes Strongest points of cell adhesion that provide mechanical binding Most abundant in tissues that are exposed to mechanical stress. (Epidermis of the skin,heart muscle) Cell to cell binding depends on cadherin family of proteins called desmoglein,desmocollin Desmosomes They contain plaque shaped structures on the cytoplasmic face of the junction which provide attachment sites for intermediate filaments ( Plaque proteins are: Plakoglobins,desmoplakins,plakophilins) Desmosome.Two types of cadherins (desmoglein and desmocolin) link adjacent cells together. Cytoplasmic plaque (desmoplakin, plakoglobin and plakophilin) link the cadherins.  DESMOSOME 1. Cell membrane 2. Cell Membrane Intermediate Desmoglein filaments Desmoplakin Plakoglobulin Desmocollin Plakophilin Outside of cell Desmoglein and desmocollin bind via anchor protein to intermediate filaments Desmosomes strongest points of cell adhesion give the tissues mechanical strength Desmosomes are found in many tissues especially abundant in skin,(epidermis) heart muscle, the neck of the uterus. DESMOSOME The importance of desmosome junctions is demonstrated by some of the skin autoimmune disease PEMPHIGUS. Affected individuals make antibodies against their own desmosomal cadherins  These antibodies bind to and disrupt the desmosomes that hold their skin epithelial cells together. This results in a severe Desmoglein 3 blistering of the skin, with leakage of body Pemphigus vulgaris fluids into the loosened epithelium. there are several Types of intermediate filaments Depending on the cell type: keratin filaments in epithelial cells, desmin filaments in heart and muscle cells. Anchoring junctions can be subclassified according to the cytoskeletal element that is involved. Actin filament attachment sites 1.Cell-cell junction (ADHERENS JUNCTIONS) 2.Cell-matrix junctions (FOCAL ADHESIONS) Intermediate filament attachment sites 1.Cell-cell junctions (DESMOSOMES) 2.Cell-matrix junctions (HEMIDESMOSOMES) HEMIDESMOSOMES (= half-desmosomes) Hemidesmosomes resemble desmosomes morphologically Cell use hemidesmosomes to attach to the basal lamina. Cell to matrix adhesion proteins ,INTEGRINS  Hemidesmosomes have only a single dense plaque on the cytoplasmic surface of the hemidesmosome (hemi=half) that anchors loops of intermediate filaments. Schematic model of hemidesmosome connecting an epithelial cell to the basal lamina  The extracellular domains of the integrins bind to a laminin protein in the basal lamina, an intracellular domain binds to an anchor protein (plectin) binds to keratin intermediate filaments. İntegrin (α6β4) and type XVII collagen (also called BPAG2) attach to the basal lamina. INTEGRIN  HEMIDESMOSOMES In a blistering skin disease called bullous pemphigoid, autoantibodies attack type XVII collagen. Mutations in plectin cause skin blisters associated with late- onset muscular dystrophy. A disease of hemidesmosomal molecules: Bullous pemphigoid III-Channel-forming junctions (Communicating junction) 1-Gap junctions  1-GAP JUNCTIONS Many cells within the tissues are not independent units Most cells in animal tissues are in communication with their neighbours via gap junction, gap junctions occur within almost all tissues (epithelial, smooth muscle,cardiacmuscle,lens ,pancreas,liver ,kidney etc. Except for a few highly differentiated cells such as skeletal muscle cells and  blood cells  1-GAP JUNCTIONS Physiologic experiments demonstrated that Fluorescent dyes can pass into the neigbouring cells (1958) It is first described by Karnowsky (1967) Gap junctions are responsible for free interchange of ions and larger molecules LM invisible EM visible Plasma membranes of adjoining cells are separated by a narrow intercellular space or gap 20 A⁰ wide.(regular separation:gap)  GAP JUNCTIONS In freze fracture replicas it has the form of plaques. large amounts of 80 A⁰ globular particles are seen At high magnification on each particle a minute central pore can be identified  Gap junctions consist of a number of tube like protein structures Proteins extend trough the lipid bilayer and project into the intercellular gap where joined to a particle in the opposing membrane. End to end bonding of these pipe like proteins forms a hydrophilic channel of 15-20 A  GAP JUNCTIONS CONSIST OF ASSEMBLIES OF SIX CONNEXINS Each pipe is composed of 2 cylindrical proteins called connexons. Connexon is composed of 6 protein subunits;connexins Permeability of Gap junctions Ions Ca Amino acids Nucleotides Sugars Cyclic AMP Can pass through the channels. Spread information along cells of a tissue. Decrease in pH and increase in Ca ions decrease the permeability. Gap junction channels allow to pass ions (to establish electrochemical continuity between the cells), small intracellular signaling molecules called second messengers e.g.cAMP (to establish a common network of information), metabolites (to allow sharing of nutrients) Gap junctions serve as direct connections between the cytoplasm of adjacent cells. Gap junctions between mammalian cells permit the passage of molecules 1.2 nm in diameter. The permeability of gap junctions is regulated by posttranslational modification of connexins (e.g. phosporylation) and environmental changes such as the cytosolic pH or the cytosolic concentration of free Ca2+ Decrease in pH and increase in Ca ions decrease the permeability of gap junction  The permeability of gap junctions is rapidly (within seconds) and reversibly reduced by experimental manipulations that decrease the cytosolic pH or increase the cytosolic concentration of free Ca2+ to very high levels.  The sharing of small metabolites and ions provides a mechanism for the coordination of function in individual cells of a tissue For example; in the heart, gap junctions rapidly pass ionic signals among muscle cells. Thus contraction of cardiac muscle cells coordinate.  Chanelles are not continually open They can open and close in response to changes in the cell. Closure of connexons of dying cells prevents the loss of nutrients from adjacent healthy cells. It is closed by rotation of six connexin subunits about a central axis  Mutations in connexin genes cause human disease Recessive mutations in the connexin-26β2, gene are the most common causes of inherited human deafness. Connexin-26 participates in the transport of K+ in the epithelia supporting the sensory hair cells in the ear. Cataract, Heart malformations (Cx43, Cx46, and Cx50/α8 )  1-They are the site of firm adhesion of cells 2-principal and possibly only type of junction that allows free interchange of substances 3-Have an Important role in the regulation of intrauterine development and differentiation It is important in the coordination of function among groups of cell Anchoring Junctions Cell-Matrix molecules  Hemidesmosomes  Focal adhesion Hemidesmosomes have only a single dense plaque on the cytoplasmic surface of the hemidesmosome (hemi=half) that anchors loops of intermediate filaments INTEGRIN The extracellular domains of the integrins bind to laminin protein in the basal lamina, an intracellular domain binds to an anchor protein (plectin) binds to keratin intermediate filaments. Schematic model of hemidesmosome connecting an epithelial cell to the basal lamina  HEMIDESMOSOMES In a blistering skin disease called bullous pemphigoid,  autoantibodies attack type XVII collagen. within the basal lamina Mutations in plectin cause skin blisters. FOCAL ADHESIONS Bind the cells to the extracellular matrix cell to matrix adhesion proteins; integrins responsible for the binding to the matrix cytoplasmic domain of the integrin binds indirectly to actin filaments. FOCAL ADHESIONS Integrin’s extracellular domains bind to components of extracellular matrix, while the cytoplasmic tail of the  subunit binds indirectly to actin  Anchoring junctions can be subclassified according to the cytoskeletal element that is involved. Actin filament attachment sites 1.Cell-cell junction (ADHERENS JUNCTIONS) 2.Cell-matrix junctions (FOCAL ADHESIONS) Intermediate filament attachment sites 1.Cell-cell junctions (DESMOSOMES) 2.Cell-matrix junctions (HEMIDESMOSOMES)  ADHESION PROTEINS 1. Cadherins (Cell-cell adhesion) Ca+2 dependent adhesion 2. Integrins Cell-matrix adhesion Other superfamilies of cell-cell adhesion proteins 3. Immunoglobulin(lg)-super Family members. N-CAM, I-CAM, VE-CAM (cell-cell adhesion) (1-7 immunoglobulin like domains ) Ca+2 independent adhesion 4. Selectins Mediate Transient Cell-Cell Adhesions in the Bloodstream. Control the binding of white blood cells to the endothelial cells lining blood vessels, Cadherin Integrin 3-immunoglobulin super family adhesion molecules (Ig-CAM) (1-7 immunoglobulin like domains ) disulphate bonds SELECTINS (A)P-selectin structure. (B) Selectins and integrins mediate cell to cell adhesion during migration of leucocytes from blood circulation to the underlying tissues. Selectins Mediate Transient Cell-Cell Adhesions in the Bloodstream Selectins are cell-surface carbohydrate-binding proteins (lectins)  The selectin family is composed of three members:  L- (leukocyte),  E- (endothelial), and  P- (platelet) selectin. Adhesive binding is calcium dependent. Ligands for selectin include so-called sialyl-Lewis X saccharides, and the best characterized counterreceptor is a mucin Like glycoprotein (GP), P-selectin GP ligand-1, present on leukocytes. Selectins have a major physiologic role in initiating the adhesion of leukocytes and platelets during the infl ammatory and hemostatic responses. L-Selectin is also a “homing receptor” that mediates lymphocyte binding to endothelium in peripheral lymph nodes. MED100-I MEDICAL BIOLOGY  Extracellular Matrix (ECM) Assoc. Prof. Özlem KURNAZ GÖMLEKSİZ E-mail: [email protected] What is the Extracellular Matrix? Give an example… Extracellular Matrix (ECM) Alt Başlık EXTRACELLULAR MATRIX All tissues consist of two components, a cellular component an extracellular component. comprises a variety of specialized structures that constitute the ECM. Molecular components are secreted and to some extent assembled by the cells of the tissue.  The matrisome is the ensemble of proteins that compose the ECM itself and associated proteins that covalently modify (e.g., chemically cross-link, phosphorylate, cleave) the ECM.  most animal cells in tissues are embedded in an ECM fills the spaces between cells and binds cells and tissues together. There are several types of extracellular matrices, consist of a variety of secreted proteins and polysaccharides. One type of extracellular matrix is exemplified by the thin, sheetlike basal laminae, previously called basement membranes, upon which layers of epithelial cells rest  proteins and polysaccharides in the ECM associated with the plasma membrane. Glycocalyx on the plasma membrane of endothelial cells or the apical membrane of intestinal epithelial cells as a polysaccharide rich coat  Matrix Structural Proteins tough fibrous proteins embedded in a gel-like polysaccharide ground adhesion proteins tendons contain a high proportion of fibrous proteins, whereas cartilage contains a high concentration of polysaccharides that form a firm compression-resistant gel. In bone, the ECM is hardened by deposition of calcium phosphate crystals. The sheetlike structure of basal laminae results from a matrix composition that differs from that found in connective tissues.  Proteoglycans, a group of glycoproteins cushion cells and bind a wide variety of extracellular molecules Collagen fibers, provide structural integrity, mechanical strength, and resilience Soluble multi-adhesive matrix proteins, such as laminin and fibronectin, bind to and cross-link adhesion receptors and other ECM components Collagen Is the Most Abundant Protein in the Extracellular Matrix is secreted mainly by fibroblasts accounts for up to 30% of the total proteins in human bodies. collagen comprises a family of 28 genetically distinct proteins. the principal structural elements of all connective tissues. are characterized by the presence of a repeated sequence of three amino acids-a tripeptide; glycine-X-Y, where X and Y are commonly proline; or hydroxyproline. All collagens are trimers in which at least some and often most of the protein chains are involved in forming a triple helix. The Gly-X-Y tripeptide plays a key role in the triple-helix structure Mutations in collagen genes chondroplasia, osteogenesis imperfecta, Alport syndrome, Ehlers-Danlos syndrome, and dystrophic EB, other collagen abnormalities contribute to osteoarthritis and osteoporosis.  During wound healing, remodeling of collagen needs to occur. Certain members of the matrix metalloproteinase family of enzymes are involved in the necessary degradation of collagen required for this process. These enzymes are produced by fibroblasts; inflammatory cells such as granulocytes; hypertrophic chondrocytes, osteoblasts, and osteoclasts that are involved in the remodeling of cartilage and bone.  Increased collagen cross-linking and deposition during tumorigenesis stiffens ECM, disrupts tissue morphogenesis, and promotes signaling activated by cell surface receptors binding to collagens, which enhance progression of tumors. Cancer cells secrete proteases that digest proteins of the ECM, allowing the cancer cells to invade surrounding tissue and metastasize to other parts of the body.  The amount of ECM Composition and amount of ECM differ according to the function of the tissue. Bone is calcified for strength and consists largely of ECM, enabling it to fulfill its functions of providing strength and support for soft tissues and of carrying muscle attachments to facilitate its lever function in movement. Cartilage also consists mainly of ECM, but it has very different properties from bone because it needs to provide articulation in joints, while at the same time needing to resist compression and provide a cushioning effect between hard bones. The dermis connects the epidermis to the underlying tissues and needs to provide great strength and elasticity to dissipate the stresses impinging on the skin. The basement membrane is essentially a thin supporting layer for cell attachment, but it has other specialized functions in tissues such as the kidney.  In adult organisms, the majority of extracellular matrices exhibit slow turnover; they are permanent or semipermanent in nature. They do, however, need to retain the capacity to respond to changes such as injury, for example, in the healing of fractures or wounds. Another type of matrix, the blood clot, needs to form rapidly and in the correct location in response to injury, but then needs to disperse as the injury is repaired. Modulation of the ECM is also important in angiogenesis, the generation of new blood vessels in response to injury or tumor growth. The role of the matrix is not exclusively structural; it provides the basis for signals transmitted to cells by adhesion receptors that bind to its components, and it acts as a reservoir for growth factors that also bind reversibly to its constituents.  The major structural protein of ECM  COLLAGEN, (the single most abundant protein in animal tissues) Other major bulk constituents of the ECM GLYCOSAMINOGLYCANS (GAGS)  carbohydrate chains  glycosaminoglycans (GAGs). GAGs are usually linked to proteins to form proteoglycans Glycosaminoglycans and Proteoglycans Absorb Water and Resist Compression GAGs strongly negatively charged most of the sugars bear carboxylic acid groups, and in chondroitin sulphate, dermatan sulphate, heparan sulphate, and keratan sulphate the amino sugars are commonly sulphated. These long carbohydrate chains do not fold into compact units. their negative charge attracts cations such as Na+ that are osmotically active and thus attract large amounts of water. GAGs fill large volumes of space are able to resist compressive forces such as the huge pressures that are exerted on cartilage in joints.  Hyaluronic acid (HA), a nonsulphated GAG, consists of up to 25,000 disaccharide units and is widely distributed in tissues. HA molecules can reach molecular weights of several million daltons, and a single molecule, swollen with water, can occupy a space of 107 nm3. HA is a lubricant in joints and facilitates cell migration in embryonic development and wound healing. Proteoglycans consist of sulphated GAGs covalently linked to a polypeptide chain, the core protein.  They vary enormously in size and carbohydrate composition. The largest can consist of up to 95% carbohydrate by weight, and aggrecan, a major constituent of cartilage, has a molecular weight of about 3 million Da. At the other extreme, decorin has a molecular weight of 40 kDa and a single carbohydrate chain. Already an enormous molecule in its own right, aggrecans in cartilage form giant complexes that have molecular weights of the order of 100 million Da and occupy a space of 5 ×1016 nm3. An aggrecan aggregate consists of a central core HA molecule with many aggrecan molecules joined to it laterally by means of linker proteins; the entire substructure resembles a bottle brush under the electron microscope.  Proteoglycans have several additional functions apart from their space filling and mechanical properties. For example, by binding to growth factors such as fibroblast growth factors (FGFs) or transforming growth factor-α and chemokines, they can regulate the activity/availability of these diffusible signaling molecules. Decorin is an example of a proteoglycan that has such regulatory powers is also involved in the formation of collagen fibers through its ability to bind to collagen. Another proteoglycan called perlecan is a critical component of the basement membrane of the kidney where its properties contribute to the filtration of plasma. Some proteoglycans are transmembrane proteins rather than components of the ECM. syndecans are integral membrane proteoglycans that contribute to the adhesive properties of focal contacts.  Elastin and Fibrillin Provide Tissue Elasticity tissues require considerable elasticity, the ability to return to normal shape after being disturbed. This property is particularly important in the skin, lungs, and blood vessels. Tissue elasticity resides largely in a network of elastic fibers that are interwoven with collagen fibers. The principal components of elastic fibers are the proteins elastin and fibrillin. Elastin is the major component and constitutes up to 50% by weight of large arteries.  It contains a series of hydrophobic domains that are responsible for its elastic properties and α- helical linker segments, rich in lysine, that are involved in forming cross-links to adjacent molecules. The resulting ECM complex consists of a network that confers on the fibrils five times the extensibility of an elastic band of the same size. Elastic fibers are covered with a sheath of 10-nm diameter microfibrils composed of the protein fibrillin. These are important for fiber assembly.  Mutations in the fibrillin gene  Marfan syndrome  the integrity of elastic fibers is compromised leading to a rupture of the aorta in severe cases. Mutations in the elastin gene cause narrowing of major arteries. Elastin overproduction in vascular walls could contribute to the pathogenesis of atherosclerosis. Elastolytic enzymes, such as aspartic protease and MMPs, degrade elastic fibers, which release elastic peptide fragments, such as Gly-Val-Ala-Pro-Gly (VGVAPG). VGVAPG peptides serve as chemotaxin to recruit monocytes and fibroblasts during development of vascular diseases and cancers, resulting in vascular intimal thickening and tumor progression.  Fibronectin Is Important for Cell Adhesion Fibronectin  non-collagenous ECM proteins play a role in regulating cell adhesion and cell behavior. less abundant in cultures of certain tumor cells than those of normal cells, it might contribute to the lower adhesiveness and metastatic properties of tumors. important in embryonic development where it provides a substratum for guiding gastrulation movements and the migration of neural crest cells. a soluble form of fibronectin is abundant in blood plasma, to contribute to blood clotting, wound healing, and phagocytosis.  Fibronectin a dimer two similar or identical protein chains, each about 200 kDa molecular weight, that are linked together near their COOH termini by two disulfide bonds. The majör structural element of these chains is the barrel-like fibronectin type III repeat. Distributed along the chain are various sites for interaction with other molecules including domains for heparin, collagen, cell binding, and self-association. The major cell-binding site consists of a tripeptide sequence (Arg-Gly-Asp, or RGD in single letter amino acid code) that is present on an exposed loop extending from one of the type III repeats. This is the site for binding α5ß1 integrin, the principal cellular fibronectin receptor. RGD sequences have subsequently been discovered in other matrix protein, for example, the blood clot protein fibrinogen. Snakes produce an RGD-containing protein, disintegrin, in their venom to prevent blood clotting, and drugs based on RGD peptides have been developed as anticlotting agents.  Fibrin A major ECM component of blood clots forms an elastic network to which cells and other ECM components bind. Polymerization of fibrin to form the network occurs when its precursor molecule fibrinogen is cleaved by the enzyme thrombin. Fibrinogen molecules are elongated structures 45 nm in length two sets of αα, Bß, and γ chains linked by disulfide bonds.  Fibrin has binding interactions with a variety of extracellular components fibronectin and heparin, the growth factors FGF-2 and vascular endothelial growth factor, and the cytokine interleukin-1. It also has binding sites for a number of CAMs including vascular endothelial (VE)-cadherin, the platelet integrin αIIbβ3, and the leukocyte integrin αmβ2 (Mac-1). These are important for promoting angiogenesis, incorporating platelets into the developing thrombus, and recruiting monocytes and neutrophils, respectively.  clots need to disperse when their function is no longer required, or if they form inappropriately. The dispersal process called fibrinolysis is mediated by an enzyme called plasmin that cleaves fibrin. Plasmin is activated by cleavage of a precursor protein plasminogen, through the action of another enzyme, tissue plasminogen activator, which binds to fibrin. Plasminogen Plasmin TPA Von Willebrand Factor a key role in the major response of platelets to vascular injury by mediating the initiation and progression of thrombus formation. Blood flow produced substantial shear forces at the blood vessel wall, and these forces oppose cell adhesion. vWF forms a bridge between collagen in the vessel wall and blood platelets sufficient to enable cell adhesion to develop.  vWF multimers are synthesized intracellularly and stored in Weibel–Palade bodies in endothelial cells and a-granules in platelets and megakaryocytes (large cells that give rise to platelets). Some vWF is secreted constitutively by endothelial cells, giving a residual plasma concentration of this protein. Regulated secretion by endothelial cells and platelets occurs in response to vascular injury. The vWF monomer has binding sites for collagen and the platelet adhesion receptors GPIb (part of the GPIb-IX-V complex) and GPIIbß3, as well as for collagen and the blood-clotting protein factor VIII. Thus, the multimeric complexes are literally strings bristling with binding sites. The larger the multimers,the more effective they are at promoting thrombus formation.  Initial platelet adhesion is mediated by the binding of GPIb on the platelet to vWF, which is, in turn, bound to collagen. This attachment is easily broken and does not result in firm adhesion. It is possible that binding between selectin molecules on the platelet surface and the carbohydrate chains on vWF also contribute to initial adhesion. GPIb-vWF interaction generates an intracellular signal within the platelets that involve changes in intracellular Ca2+ concentration and the signaling enzyme protein kinase C. The function of these signals is to bring about activation of the platelet integrin GPIIbß3 that then mediates firm adhesion to vWF. Because it has multiple binding sites for platelet adhesion molecules, vWF can also mediate platelet aggregation by bridging between them.  Platelets also adhere to fibrinogen and fibrin, other important participants in the clotting process. It appears that vWF and fibrin have complementary roles in thrombus formation. Thus, vWF mediates rapid thrombus formation at high shear rates in the absence of fibrinogen, but the thrombi are unstable. In the presence of fibrinogen thrombus development is slower but more stable. Thus, patients having congenital defects in either vWF or fibrinogen have clotting disorders.  As seen with fibrin, the process of thrombus formation by vWF requires regulation. extracellularly by a plasma enzyme called ADAMTS13, which cleaves vWF multimers to restrict their size and possibly to prevent excessive thrombus formation. The normal function of ADAMTS13 is clearly important because mutations in the gene for this enzyme cause a disease called chronic relapsing thrombocytopenic purpura. It is important to understand the mechanisms involved in thrombus their abnormal function cause thrombocytic diseases such as stroke, coronary thrombosis, phlebitis, and phlebothrombosis.  Some patients with von Willebrand disease also have symptom of gastrointestinal vascular malformations, which leads to digestive tract bleeding in these patients. As a key regulator of hemostasis, VWF predominantly inhibits blood vessel formation. Hyperactivated VEGFR-2 signaling found in vWF-deficient cells indicates that vWF may control angiogenesis by maintaining VEGF signaling at physiological levels, thereby preventing formation of unstable, fragile, and leaky vessels due to dysregulated VEGFR-2 signaling. Stem Cell Interaction With ECM Is Critical for Cell Stemness and Plasticity Stem cell renewal and differentiation are regulated by their local microenvironment, known as niche, in which ECM is a key component. Interactions between stem cells and ECM influence the stem cell migration, proliferation, survival, and differentiation. ECM stiffness and topography have a role in controlling stem cell fate. The spatial distribution of the ECM components participate in determining division axis orientation of epithelial stem cells, by remodeling the actin cytoskeleton. A stem cell with elongated shape is less likely to initiate differentiation than if it is in a circular island shape. Spreading MSCs tends to differentiate into osteoclasts and chondrocytes, while MSC rounding favors adipogenesis.  Stem cell-ECM interactions are largely mediated by integrins. Integrin interactions with the ECM, along with signals initiated by mechanical force and other ECM receptors within the niche, are critical for balancing stem cell self-renewal and differentiation. Binding of integrin to basement membrane constituents (e.g., laminin, fibronectin, and collagen IV) promotes asymmetric cell division in many stem cell types. The integrin a6/ITGA6/ CD49f, which binds to laminin, is expressed in multiple adult stem cells types and is recognized as a marker for different cancer stem cells. lntegrin-mediated mechanotransduction senses biophysical cues from ECM and modulates cellular responses by altering cytoskeleton and changing gene transcription.  MED100-I Medical Biology Structure and Function of the Endoplasmic Reticulum Molecular mechanisms of protein synthesis in Rough Endoplasmic Reticulum Assoc. Prof. Dr. Özlem KURNAZ GÖMLEKSİZ  Endoplasmic Reticulum - endoplasmic - “within the cytoplasm” - reticulum - Latin for a “a little net” - extensive network of folded membranes that extends from the nuclear envelope to which it is connected, throughout the cytoplasm  Endoplasmic Reticulum a netlike labyrinth of branching tubules and sacs that extends from nucleus to plasma membrane Sacs (cisternae), internal space, ER lumen Tubules The sacs and tubules are all interconnected by a single continuous membrane  Endoplasmic Reticulum Intracellular channels The endoplasmic reticulum serves many general functions, including the folding of protein molecules in sacs called cisternae and Intracellular transport the transport of synthesized proteins in vesicles to the Golgi Lipid biosynthesis apparatus Protein biosynthesis  There are two basic kinds of endoplasmic reticulum 1- Rough (granular ) ER 2- Smooth (agranular) ER (RER or GER) (SER) The surface of the RER is studded with ribosomes giving it a "rough" appearance The quantity of RER and SER in a cell can interchange from one type to the other, depending on changing metabolic needs.  Microsomes RER and SER regions of ER can be seperated by centrifugation. When tissue distrupted by homogenization the ER breaks into many small (100-200 nm in diameter) closed vesicles called microsomes. Microsomes are useful for studying of ER functions in vitro.  Smooth ER (SER): Regions of ER that lack bound ribosomes are called smooth endoplasmic reticulum, *Tubular or vesicular in form *SER membranes arise from GER *SER is not involved in protein synthesis *In the different cells the function of SER are different The SER functions SER has different functions in the specialized cells 1- biosynthesis of lipids 2- lipid transport 3- biosynthesis of steroid hormones 4- the metabolic reactions in the liver cells 5- contraction process in the muscle cells 6- regulation in neuronal synapse 1- SER is involved biosynthesis of lipids The synthesis of fatty acids and phospholipids occurs in the smooth ER The ER also produces cholesterol SER is the principle site of production of lipoprotein particle in liver The enzymes that synthesize the lipid component of lipids are located in the SER membrane 2- SER functions in lipid transport Dietary lipids breakdowns into fa and mg by pancreatic lipase in the small intestine absorptive epithelial cells; monoglycerides , fatty acids absorbtion-pinocytosis intestine cell SER (triglycerides) Acyl-CoA synthetase, acyltransferases Golgi Complex (apolipoproteins chylomicron )) lymphatic or blood vessels fats are mainly digested in the small intestine by pancreatic lipase the bile salts emulsifies them. That is, they break the big droplet into many smaller ones. Helps to absorbtion Complete digestion of fat (a triglyceride) results in fatty acid and monoglycerol molecules the mechanism of lipid absorption: - emulsification, - lipolysis, - micellar formation, - membrane translocation, - intracellular resynthesis, - chylomicron formation, - lymphatic drainage. 3- SER functions in biosynthesis of steroid hormones: It contains some of the enzymes required for steroid synthesis and they are abundant in ; *Leydig’s cells of testes: testesteron *Adrenal cortex cells: corticosteroids *Corpus luteum cells of ovarium: progesteron *Leydig’s cells adrenal cortex  4- SER is involved in the metabolic reactions in the liver cells (SER is abundant in hepatocytes) A- SER contains specific enzymes to detoxify (to break down) drugs, alcohol, steroid hormones and toxic chemicals; cytochrome P450 enzymes It is also called CYP enzymes The CYP enzymes catalyze the oxidation of organic substances. CYPs are the major enzymes involved in drug metabolism. Smooth ER plays a large part in detoxifying a number of organic chemicals converting them to safer water-soluble products. Human CYPs Humans have 57 genes cytochrome P450 genes 4- SER is involved in the metabolic reactions in the liver cells B- it is involved in the breakdown of glycogen into glucose Smooth ER also contains the enzyme glucose-6-phosphatase which converts glucose-6-phosphate to glucose, (a step in gluconeogenesis) The liver is also the main site in the body for gluconeogenesis Glycogen SER  5- SER participates in the contraction process in muscle cells A special type of smooth ER is found in smooth and striated muscle called “Sarcoplasmic reticulum;SR” The SR consists of a branching network of SER cisternae surrounding each myofibril  The sarcoplasmic reticulum stores and pumps calcium ions, specifically regulates Ca+2 flow in muscle cells SER membrane contains Ca+2 - ATPase pumps Ca+2 regulate muscular contraction The SR's release of Ca+2 upon electrical stimulation of the cell plays a major role in excitation-contraction coupling when the muscle is exitated, Ca+2 diffuse out of the SR in the resting state, most of the Ca+2 reside in the SR 6- Regulates neuronal synapse “a synapse is a structure that permits a neuron to pass an electrical or chemical signal to another cell.” In neuron, the end of the axon (synaptic terminal) contain a number of SER vesicles. during synapse SER is involved to Ca+2 regulation similar to myocytes. GRANULAR ER (GER) Rough ER (RER)  GER is involved in protein synthesis GER prominent in cells for protein secretion. In light microscope, it can be detected by staining with basic dyes (e.g. Nissl bodies in motor neurons) GER Nissl bodies: GER+ Ribosome clusters  In electron microscope, GER appears as parallel membrane limited flattened sacs or cisternae The membranes of the ER are continuous with the outer membrane of the nuclear envelope.  GER is particularly well developed in protein secreting cells in the digestive enzyme producing cells of the exocrine pancreas; aciner cells in the collogen and elastin producing cells of the connective tissue; fibroblast in the antibodies producing cells; plasma cell in the neurotransmitter producing cells; motor neurons in the phagocytic cells containing lysosomal enzymes ; macrophages Liver cell Plasma cell Aciner cell the rough ER in a pancreatic exocrine cell that The hepatocyte is a cell in the body that manufactures makes and secretes large amounts of digestive serum albumin, fibrinogen, and the prothrombin group of enzymes every day. The cytosol is filled with clotting factors closely packed sheets of ER membrane studded with ribosomes The GER has a central role in protein biosynthesis and their transportation within the cell  How do the right proteins get to the right places? The mechanism of the protein synthesis in GER is explained with SIGNAL HYPOTHESIS The signal hypothesis, formulated by Günter Blobel and David Sabatini in 1971, and elaborated by Blobel and his colleagues between 1975 and 1980 SIGNAL HYPOTHESIS Günter Blobel, (Nobel Prize for Medicine - 1999) 1999 Nobel Prize in Physiology has been awarded to Günter Blobel (the Rockefeller University,) for ''the discovery that proteins have intrinsic signals that govern their transport and localization in the cell''.  SIGNAL HYPOTHESIS Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific subcellular location or exported from the cell for correct activity. This phenomenon is called protein targeting (signal hypothesis) Protein targeting is necessary for proteins that are destined to work outside the cytoplasm This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases. Sorting or translocation can occur as 1. CO TRANSLATIONAL TRANSLOCATION Synthesized protein is transferred to an SRP receptor on the endoplasmic reticulum (ER). There, the nascent protein is inserted into the translocation complex 2. POSTTRANSLATIONAL TRANSLOCATION Even though most proteins are co translationally translocated, some are translated in the cytosol and later transported to their destination. This occurs for proteins that go to a mitochondrion, a chloroplast, or a peroxisome co-translational targeting (secretory pathway): ER Golgi lysosomes plasma membrane secreted proteins post-translational targeting: nucleus mitochondria Peroxisomes  According to this hypothesis; Three different types of the protein that are the related to the GER are synthesized in cells. 1- Proteins that are secreted from the cell 2- Lysosomal enzymes 3- Plasma membrane glycoproteins  In cells, molecular labels (often, amino acid sequences) are used to "address" proteins for delivery to specific locations. A characteristic feature of these targeting pathways (with the exception of cytosolic and nuclear proteins) is the presence of a short amino acid sequence at the amino terminus of a newly synthesized polypeptide called the signal sequence or signal peptide. In 1975, George Palade, at the Rockfeller Institute in New York, demonstrated that proteins with these signal sequences are synthesized on ribosomes attached to the ER membrane. Targeting signals are the pieces of information that enable the cellular transport machinery to correctly position a protein inside or outside the cell. In the absence of targeting signals, a protein will remain in the cytoplasm.  If an mRNA lacks a signal sequence protein will be synthesized entirely in the cytoplasm on free ribosomes as a structural protein of the cell  If an mRNA contains “signal sequence” initially mRNA binds to the free ribosomes in the cytoplasm, the signal peptide is synthesized on the ribosome in the cytoplasm.  The signal peptide is about 16-20 amino acids and it appears at the beginning of the polypeptide chain.  As the signal peptide emerges from the ribosome it is recognized by a special molecule in the cytosol (signal recognation particles; SRP) (SRP;6 proteins +7S RNA molecule) takes the ribosome to the ER.  SRP binds to the signal peptide and ribosome SRP binding sites; 1- signal peptide then “SRP and ribosome complex” attaches to the 2- ribosomal A site ER at specific sites called SRP receptor 3- SRP receptor on ER For transport of a polypeptide into the ER lumen, the signal sequence attaches to the SRP receptor. The hydrophobicity of the signal sequence is postulated to be the molecular key for the polypeptide's interaction with the ER membrane, which is also a hydrophobic structure. The second recognition site, ribosome receptor, serves to anchor the organelle (ribosome) to the ER membrane. SRP signal peptid The interaction between the signal sequence and the ER membrane is believed to open a channel in the membrane through which the polypeptide is transported into the ER lumen. Thus, the molecular instructions for transport into the ER (in the form of a hydrophobic sequence) are furnished by the polypeptide. SRP receptor ER lumen  SRP binds to the signal peptide and ribosome (A site) (At this moment protein synthesis is arrested) SRP and ribosome complex attaches to the ER at the specific sites called SRP receptor (or docking protein)  Upon binding to the docking SRP receptor protein, SRP is released from ribosome, GER lumen returns to cytosol it may participate in other SRP round of protein synthesis cycle Signal peptide ribosome  on the ER membrane there are pore proteins (Translocator proteins; translocon ), ribosomal large subunits attaches to this pore proteins. translocon; Sec61p complex When SRP is released from ribosome, at this moment protein synthesis starts, The signal peptide and the growing polypeptide chain is released through the translocon into the cisterna (lumen) of GER  The signal peptide is cleaved from the polypeptide chain by signal peptidase into the ER lumen. The last step; when the protein synthesis is complated the ribosomes detaches from the GER membrane, then mRNA and ribosomes may participate in an other round of protein synthesis.  Original figures The signal hypothesis as it was proposed by Günter Blobel and David Sabatini in 1971  Post-Translational Modifications in the Rough ER Newly synthesized polypeptides in the membrane and lumen of the ER undergo principal modifications before they reach their final destinations: 1. Specific proteolytic cleavages 2. Addition and processing of carbohydrates 3. Proper folding 4. Assembly into multimeric proteins 1- Specific proteolytic cleavages The signal peptide is removed from the polypeptide by the SIGNAL PEPTIDASE (protease) 2- Addition and processing of carbohydrates GLYCOSYLATION Many secreted and membrane proteins contain covalently attached carbohydrates (CH) Addition of CH chains starts in ER , terminates in Golgi Complex  Precursor oligosaccarides (OlSc )are synthesized in the cytosol, then imported into the ER or Golgi lumen by Dolichol (special lipid molecule) which holds OlSc chains in ER membrane (flip-flop )  CH (oligosaccarides) The precursor ofchains arechains the CH boundattached to specific to site the on ER the polypeptide; membrane by a dolichol lipid carrier (phospholipid) They are“Asparagine-X transferred to-serin/threonin” the polypeptide. This process is catalyzed by various oligosaccharyl transferases 3- Proper folding: translocated polypeptide chains fold and assembled in ER lumen Each polypeptide has a different folding pathway, dictated by its sequence, The folding of many newly made proteins within the GER is facilitated by some folding catalysts; 1- BIP; Binding protein assists protein folding the Sec61 channel, and another chaperon within the ER called BiP is required to pull the polypeptide chain through the channel and into the ER 2-Folding proteins; Calnexin and Calreticulin (lectins) 3- Disulfide bonds; disulfide isomerase  Formation of disulfide bonds (-s-s-) Disulfide bonds (DBs) help stabilize the tertiary and quaternary structure of many proteins (e.g. insulin) In eukaryotic cells, DBs are formed in the GER, but not in the cytosol Protein disulfide isomerase ,an enzyme localized to GER lumen, catalyzes the rearragement of disulfide bonds. DBs are common in secretory proteins and exoplasmic domains of membrane proteins, but are absent from soluble cytosolic proteins  Only properly folded proteins are transported Proteasome pathway from the GER to the Golgi Abnormally folded or unfolded proteins are exported from ER then they are degraded in the ubiquitin- proteasome pathway in the cytosol Otherwise improperly folded proteins are retained in the GER and result in ER stress,  Proteins can be translocated into the ER either during their synthesis on membrane-bound ribosomes (cotranslational translocation) or after their translations has been completed on free ribosomes in the cytosol (posttranslational translocation) Soluble proteins Plasma membrane glycoproteins  Soluble proteins destined for the ER lumen, for secretion, or for transfer to the lumen of other organelles pass completely into the ER lumen.  Plasma membrane glycoproteins synthesis Transmembrane proteins destined for the ER or for other cell membranes are translocated to the ER membrane and remain anchored there by one or more membrane-spanning a-helical regions in their polypeptide chains. Co-translational protein insertion In mammalian cells, most proteins enter the ER co- translationally Plasma membrane glycoproteins synthesis Co-translational protein insertion Topologies of the integral membrane proteins synthesized on the rough ER  Plasma membrane glycoproteins synthesis Post-translational protein insertion It was discovered that many proteins can enter ER membranes posttranslationally, that is after much or all of their synthesis is complete. ATP is required for this process The hydrophobic portions (signal pepdides) of the protein can act either as start-transfer or stop-transfer signals during the translocation process.  When a polypeptide contains multiple, alternating start-transfer and stop- transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein.  Export of proteins from the ER to Golgi complex for the further modifications * newly synthesized proteins travel along the secretory pathway in transport vesicles, which bud from the membrane of one organelle (ER) and then fuse with the another organelle membrane (Golgi).  Many resident ER proteins participated in the ER functions (e.g BIP) can escape from ER. they are returned to the ER from the Golgi apparatus The ER resident proteins contain short aa sequences (signal) called the KDEL sequences  The Golgi complex has KDEL receptors Golgi traps the ER proteins and selectively transports back to the ER.  ER Stress Various conditions can disturb ER functions, including inhibition of protein glycosylation, reduction of formation of disulfide bonds, calcium depletion from the ER lumen, impairment of protein transport from the ER to the Golgi, expression of malfolded proteins. Such ER dysfunction causes proteotoxicity in the ER, termed "ER stress” In mammalian cells, ER stress occurs under many conditions; pharmacological chemicals, decreases in oxygen, glucose, ATP and calcium ions, nutrient deprivation, developmental processes, genetic mutations, pathogenic insult. In order to survive under ER stress conditions, cells have a self-protective mechanism against ER stress, the ER stress response ; unfolded protein response, UPR Numerous pathophysiological conditions are associated with ER stress-induced apoptosis including ischemia, diabetes and neurodegenerative diseases.  There are four functionally distinct unfolded protein response, UPR The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of ER. 1-Transcriptional induction of ER chaperones increases protein folding activity and prevents protein aggregation 2- Translational attenuation reduces the load af new protein synthesis and prevent further accumulation of unfolded proteins 3- The ER associated degradation (ERAD) pathway eliminates misfolded proteins by the ubiquitin-proteasome system 4- If the stress cannot be resolved, severe and prolonged ER stress extensively impairs the ER functions\ then the cell dies by apoptosis. MED100-I MEDICAL BIOLOGY  Basal Laminae Assoc. Prof. Özlem KURNAZ GÖMLEKSİZ E-mail: [email protected] Provides a Foundation for Assembly of cells into tissue a sheet-like meshwork of ECM components no more than 60–120 nm thick In columnar and other epithelia such as intestinal lining and skin, it is a foundation on which only one surface of the cells rests. In other tissues, such as muscle or fat, the basal lamina surrounds each cell.  in regeneration after tissue damage and in embryonic development. helps four- and eight-celled embryos adhere together in a ball. In the development of the nervous system, neurons migrate along ECM pathways that contain basal laminal components. In higher animals, two distinct basal laminae are employed to form a tight barrier that limits diffusion of molecules between the blood and the brain (blood-brain barrier), and in the kidney, a specialized basal lamina serves as a selectively permeable blood filter. In muscle, the basal lamina helps protect the cell membranes from damage during contraction and relaxation.  the basal lamina is important for organizing cells