Introduction to Embryonic Development PDF

Introduction to embryonic developments Introduction to Embryonic development A multicellular organism develops from a single cell (the zygote) into a collection of many different cell types, organized into tissues and organs. Development involves cell division, body axis formation, tissue and organ development, and cell differentiation (gaining a ﬁnal cell type identity). During development, cells use both intrinsic, or inherited, information and extrinsic signals from neighbors to "decide on" their behavior and identity. Cells usually become more and more restricted in their developmental potential (the cell types they can produce) as development progresses Basic processes of development 1.The number of cells must increase through division 2.Body axes (head-tail, right-left, etc.) must form 3.Tissues must form, and organs and structures must take on their shapes 4.Individual cells must acquire their ﬁnal cell type identities (e.g., neuron) Sources of information in development Broadly speaking, there are two kinds of information that guide cells' behavior: Intrinsic (lineage) information is inherited from the mother cell, via cell division. For instance, a cell might inherit molecules that "tell" it that it belongs to the neural, or nerve cell-producing, lineage of the body. Extrinsic (positional) information is received from the cell's surroundings. For instance, a cell might get chemical signals from a neighbor, instructing it to become a particular kind of photoreceptor (light-detecting neuron). During development, cells often use both intrinsic and extrinsic information to make decisions about their identity and behavior. Intrinsic (lineage) information Extrinsic (positional) information Cellular differentiation Cellular differentiation Cellular differentiation is the process where a cell changes from one cell type to another. Usually, the cell changes to a more specialized type. Differentiation occurs numerous times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. Terminal differentiation A specialized type of differentiation, known as 'terminal differentiation', is of importance in some tissues, for example vertebrate nervous system, striated muscle, epidermis and gut. During terminal differentiation, a precursor cell formerly capable of cell division, permanently leaves the cell cycle, dismantles the cell cycle machinery and often expresses a range of genes characteristic of the cell's ﬁnal function (e.g. myosin and actin for a muscle cell). Each differentiated cell type has a speciﬁc gene expression pattern that it maintains stably. The genes expressed in a cell type specify proteins and functional RNAs needed by that particular cell type, giving it the right structure and function to do its job. Cell Potency Cell potency Cell potency is a cell's ability to differentiate into other cell types. The more cell types a cell can differentiate into, the greater its potency. Potency is also described as the gene activation potential within a cell, which like a continuum, begins with totipotency to designate a cell with the most differentiation potential, pluripotency, multipotency, oligopotency, and ﬁnally unipotency. Cell potency Totipotent: A cell that can differentiate into all cell types, including the placental tissue, is known as totipotent. In mammals, only the zygote and subsequent blastomeres are totipotent, while in plants, many differentiated cells can become totipotent with simple laboratory techniques. Pluripotent: A cell that can differentiate into all cell types of the adult organism is known as pluripotent. Such cells are called meristematic cells in higher plants and embryonic stem cells in animals (Cells of blastula and gastrula). Multipotent: A multipotent cell is one that can differentiate into multiple different, but closely related cell types. Oligopotent cells are more restricted than multipotent, but can still differentiate into a few closely related cell types. Unipotent cells can differentiate into only one cell type, but are capable of self- Examples multipotent progenitor cells include: 1.Radial glial cells (embryonic neural stem cells) that give rise to excitatory neurons in the fetal brain through the process of neurogenesis. 1.Hematopoietic stem cells (adult stem cells) from the bone marrow that give rise to red blood cells, white blood cells, and platelets 1.Mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells 1.Epithelial stem cells (progenitor cells) that give rise to the various types of skin cells Examples of oligopotent stem cells include: Lymphoid stem cells: Can differentiate into various blood cells, such as B and T cells, but not red blood cells Myeloid stem cells: Can differentiate into any of the blood stem cells found in the lymphatic system Ocular surface stem cells: Found in the cornea and conjunctival cells. Bronchoalveolar duct connection cells: Can form bronchiolar epithelium and alveolar epithelium Examples of Unipotent stem cells: Germ line stem cells: Produce sperm Epidermal stem cells: Produce skin Adult muscle stem cells: Can regenerate themselves The hallmark of stem cells is that they undergo asymmetric cell division, producing two daughter cells that are different from one another. One daughter remains a stem cell, a process called self-renewal (the dividing cell "renews" itself by making a functionally identical daughter). The other daughter cell takes on a different identity, either differentiating directly into a needed cell type or going through additional divisions to make more cells. Hematopoietic stem cells (adult stem cells) Homeotic genes Homeotic genes are master regulator genes that direct the development of particular body segments or structures. When homeotic genes are overactivated or inactivated by mutations, body structures may develop in the wrong place—sometimes dramatically so! Most animal homeotic genes encode transcription factor proteins that contain a region called the homeodomain and are called Hox genes. Hox genes are turned on by a cascade of regulatory genes; the proteins encoded by early genes regulate the expression of later genes. Hox genes are found in many animals, including fruit ﬂies, mice, and humans. Mutations in human Hox genes can cause genetic disorders. Homeotic genes The homeotic transcription factors shown in the diagram above all contain a DNA- binding protein region called the homeodomain, which is encoded by a segment of DNA called the homeobox. Because they contain a homeobox, homeotic genes of this class are sometimes called Hox genes for short. Wings usually form only in the second segment of the thorax, not in the third, which instead makes small structures called halteres that help the ﬂy balance. The job of Ultrabithorax is to repress second-segment identity and formation of wings in the third segment. When Ultrabithorax is inactivated in the developing third segment due to mutations, the halteres will be converted to a second set of wings, neatly positioned behind the normal set Embryogenesis Embryogenesis 7 steps: 1. gametogenesis 2. fertilization 3. cleavage 4. blastulation 5. gastrulation 6. neurulation 7. organogenesis 1.Gametogenesis It is a process by which the diploid germ cells undergo a number of chromosomal and morphological changes to form mature haploid gametes. Animals produce gametes directly through meiosis in organs called gonads. Males and females of a species that reproduces sexually have different forms of gametogenesis: spermatogenesis (male) in testes produce sperms. oogenesis (female) in Ovary produce ova. Common terms Animal Pole: the pole (end) of the egg where yolk is least concentrated. Animal hemisphere: the hemisphere of the egg where animal pole is located. Vegetal pole: the pole (end) of the egg where yolk is the most concentrated. Vegetal hemisphere: the hemisphere of the egg where vegetal pole is located. 2.Fertilization: It is the process whereby two sex cells (gametes) fuse together to create a new individual with genetic potentials derived from both parents. Have two separate activity: 1.Combining of genes derived from the two parents. 2.Creation of new organisms. It have 4 major steps: 1. Sperm contacts the egg 2. Sperm or its nucleus enters the egg, and regulation of sperm entry. 3. Sperm and egg nuclei fuse 4. Egg becomes activated and developmental changes begin 3. Cleavage Is the process of repeated rapid mitotic cell divisions of the zygote (unicellular structure) to form the Morula (multicellular structure). During this stage the size of the embryo does not change, the blastomeres become smaller with each division. The type & pattern of cleavage differ from species to species. Continues divisions to form a ball of 32 cells called the morula. The morula continues divisions to form the hollow blastula with up to several hundred cells. 4. Blastulation The production of a multicellular blastula as a result of cleavage Blastula cells are called blastomeres. A cavity forms within the ball of the cells called the blastocoel. Blastula of frog 5. Gastrulation The morphogenetic process called gastrulation rearranges the cells of a blastula into a three- layered (triploblastic) embryo, called a gastrula, that has a primitive gut. It means rearrangement of blastula cells that transforms the blastula into a gastrula. The blastula develops a hole in one end and cells start to migrate into the hole; this forms the gastrula Characterized by cell movement. Blastocoel will gradually disappear and a new cavity is formed Gastrocoel. The gastrula is a three-layered embryo The formation of three primary embryonic germ layers – Endoderm (inner) – Mesoderm (middle) – Ectoderm (outer) The cells at the vegetal pole invaginate, initiating gastrulation. 6. Neurulation Chordates Only Neurulation is formation of a dorsal, hollow neural tube ectodermal cells ﬂatten into neural plate the center of the plate sinks forming neural groove edge of plate is elevated to form neural folds neural folds fuse and form neural tube – anterior end develops into brain – posterior end develops into spinal cord Organogenesis Organogenesis organogenesis: the formation and development of the organs of an organism from embryonic cells Organogenesis is the process by which the three germ tissue layers of the embryo, which are the ectoderm, endoderm, and mesoderm, develop into the internal organs of the organism. Organs form the germ layers through the differentiation: the process by which a less-specialized cell becomes a more-specialized cell type. During differentiation, the embryonic stem cells express speciﬁc sets of genes which will determine their ultimate cell type. For example, some cells in the ectoderm will express the genes speciﬁc to skin cells. As a result, these cells will differentiate into epidermal cells. Therefore, the process of differentiation is regulated by cellular signaling cascades. Organogenesis Cells in the ectoderm are signaled by molecules called growth factors to form the neural plate, which rolls up to form a structure called the neural tube; the neural tube will eventually develop into the brain and spinal cord. The differing expression of various genes controls the differentiation of the mesoderm into connective tissue, as well as the ribs, spine, skeletal muscle, and lungs. The endoderm forms the lining of the digestive tract, as well as the linings of all the glands that will empty into the digestive tract; it also forms a wide variety of internal organs. Ectoderm In vertebrates, one of the primary steps during organogenesis is the formation of the neural system. The ectoderm forms epithelial cells and tissues, as well as neuronal tissues. During the formation of the neural system, special signaling molecules called growth factors signal some cells at the edge of the ectoderm to become epidermis cells. The remaining cells in the center form the neural plate. If the signaling by growth factors were disrupted, then the entire ectoderm would differentiate into neural tissue. The neural plate undergoes a series of cell movements where it rolls up and forms a tube called the neural tube. In further development, the neural tube will give rise to the brain and the spinal cord. Neural tube formation: The central region of the ectoderm forms the neural tube, which gives rise to the brain and the spinal cord. Mesoderm The mesoderm that lies on either side of the vertebrate neural tube will develop into the various connective tissues of the animal body. A spatial pattern of gene expression reorganizes the mesoderm into groups of cells called somites, with spaces between them. The somites will further develop into the ribs, lungs, and segmental (spine) muscle. The mesoderm also forms a structure called the notochord, which is rod-shaped and forms the central axis of the animal body. Mesoderm: The mesoderm aids in the production of cardiac muscles, skeletal muscle, smooth muscle, tissues within the kidneys, and red blood cells. Endoderm The endoderm consists, at ﬁrst, of ﬂattened cells, which subsequently become columnar. It forms the epithelial lining of the whole of the digestive tube (except part of the mouth and pharynx) and the terminal part of the rectum (which is lined by involutions of the ectoderm). It also forms the lining cells of all the glands which open into the digestive tube, including those of the liver and pancreas; the epithelium of the auditory tube and tympanic cavity; the trachea, bronchi, and air cells of the lungs; the urinary bladder and part of the urethra; and the follicle lining of the thyroid gland and thymus. Additionally, the endoderm forms internal organs including the stomach, the colon, the liver, the pancreas, the urinary bladder, the epithelial parts of trachea, the lungs, the pharynx, the thyroid, the parathyroid, and the intestines. Steps in embryonic developments 7 steps: 1. gametogenesis 2. fertilization 3. cleavage 4. blastulation 5. gastrulation 6. neurulation 7. organogenesis 1.Gametogenesis It is a process by which the diploid germ cells undergo a number of chromosomal and morphological changes to form mature haploid gametes. Animals produce gametes directly through meiosis in organs called gonads. Males and females of a species that reproduces sexually have different forms of gametogenesis: spermatogenesis (male) in testes produce sperms. oogenesis (female) in Ovary produce ova. Common terms Animal Pole: the pole (end) of the egg where yolk is least concentrated. Animal hemisphere: the hemisphere of the egg where animal pole is located. Vegetal pole: the pole (end) of the egg where yolk is the most concentrated. Vegetal hemisphere: the hemisphere of the egg where vegetal pole is located. 2.Fertilization: It is the process whereby two sex cells (gametes) fuse together to create a new individual with genetic potentials derived from both parents. Have two separate activity: 1.Combining of genes derived from the two parents. 2.Creation of new organisms. It have 4 major steps: 1. Sperm contacts the egg 2. Sperm or its nucleus enters the egg, and regulation of sperm entry. 3. Sperm and egg nuclei fuse 4. Egg becomes activated and developmental changes begin 3. Cleavage Is the process of repeated rapid mitotic cell divisions of the zygote (unicellular structure) to form the Morula (multicellular structure). During this stage the size of the embryo does not change, the blastomeres become smaller with each division. The type & pattern of cleavage differ from species to species. Continues divisions to form a ball of 32 cells called the morula. The morula continues divisions to form the hollow blastula with up to several hundred cells. 4. Blastulation The production of a multicellular blastula as a result of cleavage Blastula cells are called blastomeres. A cavity forms within the ball of the cells called the blastocoel. Blastula of frog 5. Gastrulation The morphogenetic process called gastrulation rearranges the cells of a blastula into a three- layered (triploblastic) embryo, called a gastrula, that has a primitive gut. It means rearrangement of blastula cells that transforms the blastula into a gastrula. The blastula develops a hole in one end and cells start to migrate into the hole; this forms the gastrula Characterized by cell movement. Blastocoel will gradually disappear and a new cavity is formed Gastrocoel. The gastrula is a three-layered embryo The formation of three primary embryonic germ layers – Endoderm (inner) – Mesoderm (middle) – Ectoderm (outer) The pattern of gastrulation is affected by the amount of yolk. The cells at the vegetal pole invaginate, initiating gastrulation. 6. Neurulation Chordates Only Neurulation is formation of a dorsal, hollow neural tube ectodermal cells ﬂatten into neural plate the center of the plate sinks forming neural groove edge of plate is elevated to form neural folds neural folds fuse and form neural tube – anterior end develops into brain – posterior end develops into spinal cord Neurulation Metamorphosis 1 Metamorphosis Metamorphosis – The changes in the shape or characteristics of an organism’s body as it grows and matures. Some animals, especially insects, have one kind of body when they are young and a very different kind of body when they are adults. Metamorphosis refers to the way that certain organisms develop, grow, and change form. Two forms of development (change) INCOMPLETE METAMORPHOSIS - has THREE stages COMPLETE METAMORPHOSIS - has FOUR stages. Incomplete Metamorphosis Egg- nymph- adult *Three stages of development At each stage the insect looks much the same as it does when it becomes an adult. Examples: Grasshopper or cockroaches 3 STAGES OF INCOMPLETE METAMORPHOSIS Egg Nymph Adult EGG A female insect lays eggs. These eggs are often covered by an egg case which protects the eggs and holds them together. NYMPH The eggs hatch into nymphs. Nymphs looks like small adults, but usually don't have wings. Insect nymphs eat the same food that the adult insect eats. Nymphs shed or molt their exoskeletons (outer casings made up of a hard substance called chitin) and replace them with larger ones several times as they grow. Most nymphs molt 4-8 times. ADULT The insects stop molting when they reach their adult size. By this time, they have also grown wings. Complete Metamorphosis Four stages of development Egg -> Larvae-> Pupae-> Adult 4 STAGES OF COMPLETE METAMORPHOSIS Egg Larva Pupa Adult EGG The female lays eggs. LARVA Larva hatch from the eggs. They do not look like adult insects. They usually have a worm-like shape. Caterpillars(butterﬂy), maggots(ﬂy), and grubs(beetle) are all just the larval stages of insects. Larvae molt their skin several times and they grow slightly larger. PUPA Larva make cocoons around themselves. Larva don't eat while they're inside their cocoons. Their bodies develop into an adult shape with wings, legs, internal organs, etc. This change takes anywhere from 4 days to many months. ADULT Inside the cocoon, the larvae change into adults. After a period of time, the adult breaks out of the cocoon. Complete Metamorphosis Cell injury Cell Injury If the cells fail to adapt under stress, they undergo certain changes called cell injury. The affected cells may recover from the injury (reversible) or may die (irreversible). Causes of Cell Injury oxygen deprivation (anoxia) physical agents chemical agents infections agents immunologic reactions genetic defects nutritional imbalances Important targets of cell injury Aerobic respiration – ATP depletion or decreased synthesis. Cell membranes - plasma membranes, mitochondrial, lysosomal and other organelle membranes. Protein synthesis. Cytoskeleton. Genetic apparatus. Ischaemia/Reperfusion injury Ischaemia/Reperfusion injury If cells are reversibly injured due to ischaemia, complete recovery occurs following restoration of blood ﬂow. However, reperfusion can result in more damage including cell death. This is due to incompletely metabolised products producing reactive oxygen species on re-introduction of oxygen (especially damaging to mitochondria; loss of anti-oxidants during ischaemia; inﬂow of calcium with the renewed blood ﬂow; recruitment of leukocytes to the injured area. Reperfusion injury is especially important in ischaemic damage to the heart and brain and in organ transplantation. The importance of free radicals Free radicals have a single unpaired electron in the outer orbit. They are highly reactive with adjacent molecules. Are usually derived from oxygen to produce reactive oxygen species, superoxide, hydroxyl radicals,H2O2,etc. Protective molecules include superoxide dismutase, glutathione peroxidase, vitamin E, vitamin C, catalase. During cell injury free radicals produced in excess, they react with and damage proteins, lipids, carbohydrates, nucleic acids. These damaged molecules may themselves be reactive species with a chain reaction being set up with widespread damage. Free radicals In addition to oxygen-derived free radicals, nitric oxide (NO) can act as a free radical and be converted to an even more reactive anion. Iron and copper catalyze free radical formation and are thus important in the generation of reactive oxygen species. Free radicals cause lipid peroxidation in cell membranes, oxidation of amino acids and proteins resulting in fragmentation, and protein-protein cross linkages. Altered proteins are acted on by the proteosomes with further cell damage. Membrane damage Membrane damage Mitochondria – mitochondrial permeability transition; this non-selective pore may be reversible or become permanent leading to cell death. Leakage of cytochrome C can trigger apoptosis. Plasma membrane – mechanisms include those occuring with hypoxia/ischaemia and free radicals, but also immune mechanisms as with complement activation and perforin from lymphocyte attack on cells infected with a virus. All membranes may be damaged and ruptured by mechanical force as in trauma, or by ice crystals as in extreme cold. Damage to lysosomal membranes can lead to cell death by necrosis. Morphology of Cell Injury Reversible: Cellular swelling and vacuole formation (Hydropic changes) Changes at this stage are better appreciated by EM that may show blebbing of the plasma membrane, swelling of mitochondria and dilatation of ER Morphology of Cell Injury Irreversible/Necrosis The changes are produced by enzymatic digestion of dead cellular elements, denaturation of proteins and autolysis (by lysosomal enzymes) Cytoplasm - increased eosinophilia Nucleus - nonspeciﬁc breakdown of DNA leading to pyknosis (shrinkage), karyolysis (fading) and karyorrhexis (fragmentation). Cell Death Cell Death Death of cells occurs in two ways: Necrosis--(irreversible injury) changes produced by enzymatic digestion of dead cellular elements Apoptosis--vital process that helps eliminate unwanted cells--an internally programmed series of events effected by dedicated gene products Reasons of Cell Death Mechanisms of cell death caused by different agents may vary. However, certain biochemical events are seen in the process of cell necrosis: ATP depletion Loss of calcium homeostasis and free cytosolic calcium Free radicals: superoxide anions, Hydroxyl radicals, hydrogen peroxide Defective membrane permeability Mitochondrial damage Cytoskeletal damage Morphological Forms of Programmed Cell Death Apoptosis Apoptosis This process helps to eliminate unwanted cells by an internally programmed series of events effected by dedicated gene products. It serves several vital functions and is seen under various settings. During development for removal of excess cells during embryogenesis To maintain cell population in tissues with high turnover of cells, such as skin, bowels. To eliminate immune cells after cytokine depletion, and autoreactive T- cells in developing thymus. To remove damaged cells by virus To eliminate cells with DNA damage by radiation, cytotoxic agents etc. Cell death in tumours. Mechanisms of Apoptosis Apoptosis can be induced by various factors under both physiological and pathological conditions: It is an energy-dependent cascade of molecular events which include protein cleavage by a group of enzymes called caspases, protein cross-linking, and DNA breakdown. Apoptosis is regulated by a large family of genes some of which are inhibitory (Bcl-2) and some are stimulatory (Bax) ad Bad. Apoptosis There are a number of mechanisms through which apoptosis can be induced in cells. The sensitivity of cells to any of these stimuli can vary depending on a number of factors such as: the expression of pro- and anti- apoptotic proteins (eg. the Bcl-2 proteins or the Inhibitor of Apoptosis Proteins), the severity of the stimulus and the stage of the cell cycle. Role of mitochondria in apoptosis The anti-apoptotic bcl-2 proteins are often found in the cytosol of mitochndria where they act as sensors of cellular damage or stress. Following cellular stress bcl-2 relocate to the surface of the mitochondria where the pro- apoptotic proteins are located. This interaction between pro- and anti-apoptotic proteins disrupts the normal function of the anti- apoptotic bcl-2 proteins and can lead to the formation of pores in the mitochondria and the release of cytochrome C and other pro-apoptotic molecules from the intermembrane space. This in turn leads to the formation of the apoptosome and the activation of the caspase cascade. The release of cytochrome C from the mitochondria is a particularly important event in the induction of apoptosis. Once cytochrome C has been released into the cytosol it is able to interact with a protein called Apaf-1. This leads to the recruitment of pro- caspase 9 into a multi-protein complex with cytochrome C and Apaf-1. Caspases and Apoptosis One of the hallmarks of CAD: Caspase activated deoxyribonuclease apoptosis is the cleavage of chromosomal DNA into nucleosomal units. The caspases play an important role in this process by activating DNases, inhibiting DNA repair enzymes and breaking down structural proteins in the nucleus. PARP: Poly ADP Ribose Polymerase Morphology of Apoptosis Shrinkage of cells Condensation of nuclear chromatin peripherally under nuclear membrane Formation of apoptotic bodies by fragmentation of the cells and nuclei. The fragments remain membrane-bound and contain cell organelles with or without nuclear fragments. Phagocytosis of apoptotic bodies by adjacent healthy cells or phagocytes. Unlike necrosis, apoptosis is not accompanied by inﬂammatory reaction Autophagy When cells are faced with an inadequate supply of nutrients in their extracellular ﬂuid (ECF), they may begin to cannibalize some of their internal organelles (e.g. mitochondria) for re-use of their components. Autophagy refers to a set of diverse processes whereby intracytoplasmic material is delivered to lysosomes. Autophagy Autophagy is a regulated process for the removal of damaged proteins and organelles. Autophagy occurs under basal conditions and is stimulated by environmental factors such as starvation. There is evidence that proteins that are linked to tumorigenesis can regulate the rate of autophagy, with oncogenes in general blocking and tumour suppressors stimulating the process. The removal of damaged cellular components, especially damaged mitochondria, might decrease the level of reactive oxygen species (ROS), which in turn might reduce genomic instability or forestall cellular senescence. Such mechanisms might allow moderate increases in autophagy to reduce the incidence of cancer and prolong lifespan. Autophagy involves: formation of a double membrane within the cell which envelops the materials to be degraded into a vesicle called an autophagosome. The autophagosome then fuses with a lysosome forming an autolysosome whose hydrolytic enzymes degrade the materials. Death by Injury vs. Death by Suicide (Necrosis vs. Apoptosis) Etiology of cell death Major Factors Accidental Genetic Necrosis Apoptosis Necrosis: The sum of the morphologic changes that follow cell death in a living tissue or organ Apoptosis: a physiological process that includes speciﬁc suicide signals leading to cell death Necrosis: a pathological response Apoptosis: a physiological response to to cellular injury speciﬁc suicide signals, or lack of survival signals Chromatin condenses and migrates to nuclear membrane. Internucleosomal cleavage leads to Chromatin clumps laddering of DNA at the nucleosomal repeat length, ca. 200 bp. Mitochondria swell and rupture Cytoplasm shrinks without membrane rupture Plasma membrane lyses Blebbing of plasma and nuclear membranes Cell contents are packaged in membrane bounded Cell contents spill out bodies, internal organelles still functioning, to be engulfed by neighbours. Epitopes appear on plasma membrane marking General inﬂammatory response is cell as a phagocytic target. triggered No spillage, no inﬂammation Thank you Overview of cell signalling Cell signaling 1. Overview of cell signaling 1. Types of Signaling 1. Types of ligands 1. Types of receptors 1. Signal relay pathway Phosphorylation & Secondary Messenger 1. Overview of cell signaling Cells typically communicate using chemical signals. These chemical signals, which are proteins or other molecules produced by a sending cell, are often secreted from the cell and released into the extracellular space 1. Overview of cell signaling The message carried by a ligand is often relayed through a chain of chemical messengers inside the cell. The relay of signals inside a cell are known as intracellular signal transduction pathways. 2. Types of cell signaling There are four basic categories of chemical signaling found in multicellular organisms: 1.paracrine signaling, 2.autocrine signaling, 3.endocrine signaling, 4.Juxtacrine signaling Signaling through cell-cell contact Intracellular junctions (joining) Cell walls of plant cells perforated with channels called plasmodesma. In animals are intracellular junctions. Tight junction Desmosomes Gap junctions Adhere, interact and communicate 3.Types of Signaling Ligands A. Ligands that bind to cell-surface receptors: 1.Neurotransmitters (NT), i.e. norepinephrine, histamine 2. Peptide hormones (P), i.e. insulin - can't cross membrane 3. Growth factors (GF), i.e. NGF, EGF, PDGF 4. Lipophilic signaling molecules, i.e. prostaglandins B. Ligands that bind to intracellular receptors: lipid soluble hormones that diffuse across the plasma membrane and interact with receptors in the cytosol or nucleus. i.e. steroids, thyroxine, retinoic acid, nitric oxide. Receptors and signal relay pathways 4. Receptors 1. Intra cellular receptors 1. Cell Surface receptors 1. Intracellular receptors Ex: Receptors for Thyroid hormone, steroidal hormones Cell surface receptors / Membrane receptors A. Ion channel linked receptors B. Enzyme-linked receptors (Receptor Tyrosine Kinase) C. G-protein-linked receptors A. Ion channel linked receptors + + 2+ − Ligand-gated ion channels open to allow ions such as Na , K , Ca , and/or Cl to pass through the membrane in response to the binding of a chemical messenger (i.e. a ligand), such as a neurotransmitter. A. Ion channel linked receptors Receptors binds with ligand. (Ex:Nicotinic Receptor) Conformational change in the protein’s structure that allows ions such as Na, Ca,Mg, and H2 to pass through. Open a channel through the membrane that allows speciﬁc ions pass through. B. G-protein-linked receptors G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors G-protein-linked receptors activate a class of GTP- binding proteins (G-proteins) G proteins are molecular switches They are turned on for brief periods while bound to GTP They switch themselves off by hydrolysing GTP to G proteins Some G proteins directly regulate ion channels Others activate adenylate cyclase, thus increasing intracellular cyclic AMP Some activate the enzyme Phospholipase C, thus increasing intracellular inositol triphosphate (IP3) and Diacylglycerol (DAG) C. Enzyme- Many receptors have intracellular domains with enzyme function linked Most are receptor tyrosine-kinases (RTK) receptors They phosphorylate tyrosine residues in selected intracellular proteins These receptors are activated by growth factors, thus being important in cell proliferation Receptor tyrosine kinases Receptor tyrosine kinase activation results in assembly of an intracellular signalling complex This complex activates a small GTP-binding protein, Ras Ras activates a cascade of protein kinases that relay the signal to the nucleus Mutations that make Ras hyperactive are a common way of inducing increased proliferation in cancer 5. Signal relay pathways Signal relay pathways The message carried by a ligand is often relayed through a chain of chemical messengers inside the cell. The relay of signals inside a cell are known as intracellular signal transduction pathways. Signal relay pathways Signal relay pathways -1. Phosphorylation one of the most common tricks for altering protein activity is the addition of a phosphate group to one or more sites on the protein, called phosphorylation. A. Phosphorylation example: MAPK signaling cascade Together, Raf, MEK, and the ERKs make up three-tiered kinase signaling pathway called a mitogen-activated protein kinase (MAPK) MAPK signaling cascade To get a better sense of how phosphorylation works, let’s examine a real-life example of a signaling pathway that uses this technique: growth factor signaling. Speciﬁcally, we'll look at part of the epidermal growth factor (EGF) pathway that acts through a series of kinases to produce a cellular response. Phosphorylation (marked as a P) is important at many stages of this pathway. When growth factor ligands bind to their receptors, the receptors pair up and act as kinases, attaching phosphate groups to one another’s intracellular tails. The activated receptors trigger a series of events. These events activate the kinase Raf. Active Raf phosphorylates and activates MEK, which phosphorylates and activates the ERKs. The ERKs phosphorylate and activate a variety of target molecules. These include transcription factors, like c-Myc, as well as cytoplasmic targets. The activated targets promote cell growth and division. Together, Raf, MEK, and the ERKs make up a three-tiered kinase signaling pathway B.Signal relay pathways- Second messengers Second messengers include ions-calcium; cyclic AMP (cAMP), a derivative of ATP; and inositol phosphates, which are made from phospholipids. Second messenger Binding of ligands (ﬁrst messengers) results frequently in the production of shortlived small molecules (second messengers). The ﬁrst identiﬁed second messenger was cyclic AMP (cAMP), which regulates the activity of protein kinase A. The binding of cAMP to the regulatory subunit results in the dissociation of the inactive tetramer and activation of the catalytic subunit. Because the binding is positively cooperative, small changes in cAMP concentration are translated into large changes of protein kinase A activity. Similarly, cyclic GMP (cGMP) regulates the activity of protein Second messenger - cAMP Production: ATP converted to cAMP by adenylate cyclase Second messenger - cAMP Action: cAMP-dependent protein kinase (PKA) is a tetramer of catalytic and regulatory subunits.cAMP binding leads to Dissociation of regulatory subunits and release of catalytic Subunits which then Phosphorylate target proteins in cytoplasm: Production: ATP converted to cAMP by adenylate cyclase Cell Junctions Cell Junctions Neighboring cells in tissues, organs, or organ systems often adhere, interact, and communicate through direct physical contact Intercellular junctions facilitate this contact There are several types of intercellular junctions Plasmodesmata Tight junctions Desmosomes Gap junctions Plasmodesmata in Plant Cells Plasmodesmata are channels that perforate plant cell walls Through plasmodesmata, water and small solutes (and sometimes proteins and RNA) can pass from cell to cell Cell walls Interior of cell Interior of cell 0.5 m Plasmodesmata Plasma membranes Tight Junctions, Desmosomes, and Gap Junctions in Animal Cells At tight junctions, membranes of neighboring cells are pressed together, preventing leakage of extracellular ﬂuid Gap junctions (communicating junctions) provide cytoplasmic channels between adjacent cells Desmosomes (anchoring junctions) fasten cells together into strong sheets Cell Junctions in Animal Tissues Tight junctions prevent ﬂuid from moving Tight junction across a layer of cells TEM 0.5 m Tight junction Intermediate ﬁlaments Desmosome TEM 1 m Gap junction Ions or small molecules Space between cells TEM Extracellular Plasma membranes matrix of adjacent cells 0.1 m Structural and functional organization of cell membrane The Plasma Membrane 2 Plasma Membrane Boundary that separates the living cell from it’s non-living surroundings. Phospholipid bilayer Amphipathic - having both: hydrophilic heads Phospholipid hydrophobic tails ~8 nm thick Is a dynamic structure Schematic representations of three types of membrane lipid.(a) Phosphatidylcholine, a glycerophospholipid. (b) Glycolipid. (c) A sterol. Saturated versus Unsaturated Phospholipids Membranes are more ﬂuid when they contain more unsaturated fatty acids within their phospholipids. More unsaturated fatty acids result in increased distance between the lipids making the layer more Cholesterol and Phospholipids It is found in the cell membranes of animals but not plants. It affects the ﬂuidity of the membrane. Cholesterol Functions in 3 ways It can weakly bind to hydrocarbon tails making it more diﬃcult for smaller molecules to cross membrane. If the phospholipids are saturated, cholesterol prevents them from being packed too closely, making the membrane more ﬂuid. However, if the phospholipids are unsaturated there are kinks in the tails where the cholesterol molecules can ﬁll in and anchor them making the membrane less ﬂuid. 10 Lipids: critical role in maintaining membrane ﬂuidity Saturated fatty acids stack stiffer nicely Unsaturated fatty acids –more ﬂuid; double bond causes kinks Stacks poorly More Shorter chains – stack poorly; ﬂuid More movement Length & saturation of hydrocarbon tails affect packing & membrane ﬂuidity cholesterol – At high temperature has a loosening effect which reduces the gaps in membrane and decreases the ﬂuidity of cell membrane – At low temperature has a stiffening effect where it enhances the gaps in membrane and increases the ﬂuidity of cell membrane Cholesterol Cholesterol within the animal cell membrane Most organisms regulate membrane ﬂuidity “Homeoviscous adaptation” Fish, plants Mammals, palm trees 0-20ºC 30-37ºC Polyunsaturated Fattyacids Saturated Fattyacids Shorter chains cholesterol Longer chains cholesterol Summary 1. Fluid Mosaic Model: ﬂuid nature & asymmetric distribution of components 2. Components: Lipids – phospholipids, sterols, glycolipids Fluidity Proteins – integral, peripheral, lipid-linked transport, receptors, enzymes, structural support, electron transport, specialized functional domains Carbohydrates – as glycolipids & glycoproteins external glycocalyx The membrane is ﬂuid but also fairly rigid and can burst if penetrated or if a cell takes in too much water. The mosaic nature of the plasma membrane allows a very ﬁne needle to easily penetrate it without causing it to burst and allows it to self-seal when the needle is extracted. If saturated fatty acids are compressed by decreasing temperatures, they press in on each other, making a dense and fairly rigid membrane. If unsaturated fatty acids are compressed, the "kinks" in their tails push adjacent phospholipid molecules away, which helps maintain ﬂuidity in the membrane. The ratio of saturated and unsaturated fatty acids determines the ﬂuidity in the membrane at cold temperatures. Cholesterol functions as a buffer, preventing lower temperatures from inhibiting ﬂuidity and preventing higher temperatures from increasing ﬂuidity. Membrane proteins Integral proteins span lipid bilayer called transmembrane proteins hydrophobic regions consist of one or more stretches of nonpolar amino acids often coiled into alpha helices Role of membrane proteins Transport – channels and pumps Links to structural proteins Receptors - doorbells Enzymes – localized biochemical reactions Energy Generation – utilize gradient Six major functions of membrane proteins Transport Enzymatic activity Signal transduction Cell-cell recognition Intercellular joining Attachment to the cytoskeleton and extracellular matrix (ECM) Major functions of membrane proteins Transport. (left) A protein that spans the membrane may provide a hydrophilic channel across the membrane that is selective for a particular solute. (right) Other transport proteins shuttle a substance from one side to the other by changing shape. Some of these proteins hydrolyze ATP as an energy ssource to actively pump substances across the ATP membrane. Enzymatic activity. A protein built into the membrane may be an Enzymes enzyme with its active site exposed to substances in the adjacent solution. In some cases, several enzymes in a membrane are organized as a team that carries out sequential steps of a metabolic pathway. Signal Signal transduction. A membrane protein may have a binding site with a speciﬁc shape that ﬁts the shape of a chemical messenger, such as a hormone. The external messenger (signal) may cause a conformational change in the protein (receptor) that relays the message to the inside of the cell. Receptor Cell-cell recognition. Some glyco-proteins serve as identiﬁcation tags that are speciﬁcally recognized by other cells. Glyco- protein Intercellular joining. Membrane proteins of adjacent cells may hook together in various kinds of junctions, such as gap junctions or tight junctions Attachment to the cytoskeleton and extracellular matrix (ECM) Microﬁlaments or other elements of the cytoskeleton may be bonded to membrane proteins, a function that helps maintain cell shape and stabilizes the location of certain membrane proteins. Proteins that adhere to the ECM can coordinate extracellular and intracellular changes Extra Cellular Matrix (ECM) Extracellular components and connections between cells help coordinate cellular activities Most cells synthesize and secrete materials that are external to the plasma membrane These extracellular structures include – Cell walls of plants – The extracellular matrix (ECM) of animal cells – Intercellular junctions Cell Walls of Plants The cell wall is an extracellular structure that distinguishes plant cells from animal cells Secondary cell wall Primary cell wall Prokaryotes, fungi, and some protists also have cell walls Middle lamella The cell wall protects the plant cell, 1 m maintains its shape, and prevents Central vacuole Cytosol excessive uptake of water Plasma membrane Plant cell walls Plant cell walls are made of cellulose ﬁbers embedded in other polysaccharides and protein Plasmodesmata The Extracellular Matrix (ECM) of Animal Cells Animal cells lack cell walls but are covered by an elaborate extracellular matrix (ECM) Collagen EXTRACELLULAR FLUID Proteogl complex Fibronectin The ECM is made up of Integrins glycoproteins such as collagen, proteoglycans, and Plasma membrane ﬁbronectin Micro- CYTOPLASM ﬁlaments ECM proteins bind to receptor proteins in the plasma membrane called integrins Functions of the Extracellular Matrix (ECM) – Provide mechanical support to cells and tissues – Adhesion of cells – Movement of the cells – Regulation of the cell movement ECM Molecular components The macromolecules that constitute the extracellular matrix are mainly produced locally by cells in the matrix. In connective tissue ﬁbroblast secretes the macromolecules. ECM contains three major classes of biomolecules (i) The structual proteins, collagen, elastin (ii) Certain specialized proteins such as Fibronectin and laminin iii) Proteoglycans: Glycosaminoglycans (GAGs) Heparan sulfate, Chondroitin sulfate, Keratan sulfate ECM has been found to be involved in many normal and pathologic processes i.e. inﬂammatory states, spread of cancer cells, rheumatoid arthritis and osteoarthritis. Structure of collagen All collagen types have a triple helical structure. Mature collagen type 1, containing approximately 1000 amino acids. Each polypeptide subunit or alpha chain is twisted into a left handed helix of three residues/ turn. 3 of these α-chains are then wounded into a right handed super helix forming a rod-like molecule of 1.4 nm in diameter and about 300 nm long. A striking characteristic of collagen is the occurrence of glycine residues at every third position of the triple helical portion of the alpha chain. Collagen undergoes extensive post-translational modiﬁcation Collagen is synthesized on ribosome in a precursor form, Pre- procollagen, which contains a leader or signal sequence that directs the polypeptide chain into the lumen of ER, where the leader sequence is removed, Hydroxilation of proline and lysine residues and glycolysation of hydroxylysine in the procollagen molecules also take place at this site. Collagen It is the major component of most connective tissues, constitute approximately 25% of protein of mammals and it is the most abundant protein in the animal world. It provides extracellular framework for all animals and exists in every animal tissues. At least 25 distinct types of collagen made up of over 30 distinct polypeptide chains have been identiﬁed in human tissues. ECM: Elastin, Fibronectin, Laminin and Gags Elastin It is a connective tissue protein that is responsible for properties of extensibiity and elastic recoil in tissues. Although not as widespread as collagen, elastin is present in large amounts, particularly in tissues that require these physical properties e.g. lung, blood vessels and elastic ligaments and small amounts in ear and skin. It is synthesized as soluble monomer of 70kDa called tropoelastin, deletion of elastin gene have been found in 90% of the subjects with Williams syndrome, a developmental disorder affecting connective tissue and the central nervous system. A number of skin diseases are associated with accumulation of elastin. Alternatively, a decrease of elastin is found in – conditions such as pulmonary emphysema and aging of the skin Laminin It is a protein of about 850 kDa and 70 nm long, consists of 3 polypeptide chain linked together to form an elongated cruciform shape. It has binding sites for type IV collagen, heparin and integrins. It helps in the anchoring of the lamina to the cells. Collagen binding site Heparin binding site Fibronectin It is a major glycoprotein of ECM (MW= 600 kDa), also found in soluble form in plasma. It contains an Arg-Gly-Asp (RGD) sequence that binds to the Integrins. The protein is involved in the adhesion of cells to ECM and cell migration. The amount of ﬁbronectin around many transformed cells are sharply reduced partly explaining the faulty interaction with the ECM Proteoglycan and Glycosaminoglycans (GAG) Proteoglycans are proteins that contain covalently linked glycosaminoglycans. At least 30 have been characterized and given names such as syndecan, serglycin. They vary in tissue distribution. The protein bound covalently to glycosaminoglycan is called core proteins. The amount of carbohydrate in a proteoglycan is usually much greater than is found in a glycoprotein and many comprise up to 95% of this weight. There are at least seven glycosaminoglycans (GAGs), Hyaluronic acid, chondoitin sulfate, Keratin sulfate, chondroitan sulfate I and II, heparin, heparan sulfate and dermatan sulfate. 1. Transportation across membranes 2. Passive Transport a. Diffusion b. Facilitated Diffusion c. Osmosis Cell Transportation How things get in and out of Cells? Cell Transportation Semi- Permeability Types of Cellular Transport Weeee!!! Passive Transport cell doesn’t use energy 1. Diffusion 2. Facilitated Diffusion high 3. Osmosis low Active Transport cell does use energy This is gonna be 4. Protein Pumps hard work!! 5. Endocytosis high 6. Exocytosis low Three Forms of Transport Across the Membrane Example: Oxygen or water diffusing into a cell and carbon dioxide diffusing out. Three Forms of Transport Across the Membrane Passive Transport Solutes ﬂow down the concentration gradient * The cell does not use any energy The 3 most common types of Passive Transport are: 1. Diffusion 2. Osmosis 3. Facilitated Diffusion Passive Transport cell uses no energy molecules move randomly Molecules spread out from an area of high concentration to an area of low concentration. (High Low) Three types: Passive Transport 1. Diffusion 1. Diffusion: random movement of particles from an area of high concentration to an area of low concentration. Diffusion continues until all molecules are evenly spaced (equilibrium is reached) Note: molecules will still move around but stay spread out. Passive Transport: 2. Osmosis Osmosis: diffusion of water through a selectively permeable membrane Water moves from high water concentration to Water moves low concentrations freely through pores. Solute (green) to large to move across. Osmotic Pressure Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward ﬂow of its pure solvent across a semipermeable membrane. If there are solute molecules only in one side of the system, then the pressure that stops the ﬂow of the solutes is called the osmotic pressure. Turgor Pressure Internal pressure applied to a cell wall when water moves by osmosis out of the cell. The pressure pushes the plasma membrane against the cell wall. Plasmolysis is the loss of Turgor Pressure… therefore the cell collapses. What type of solution are these cells in? A B C Hypertonic Isotonic Hypotonic All living cells must be surrounded by Water. These water environments are classiﬁed as by the concentration of solutes in the solution. The environments are classiﬁed as: 1. Isotonic 2. Hypertonic 3. Hypotonic Dealing osmatic pressure by organisms Bacteria and plants have cell walls that prevent them from over-expanding. In plants the pressure exerted on the cell wall is called tugor pressure. A protist like paramecium has contractile vacuoles that collect water ﬂowing in and pump it out to prevent them from over-expanding. Salt water ﬁsh pump salt out of their specialized gills so they do not dehydrate. Kidneys keep the blood isotonic by remove excess salt and water. Passive Transport: 3. Facilitated Diffusion A B Facilitated diffusion: diffusion of speciﬁc particles through transport proteins found in the membrane Facilitated Diffusion a. Transport Proteins are diffusion (Lipid speciﬁc – they “select” (Channel Bilayer) only certain molecules Protein) to cross the membrane b. Transports larger or charged molecules Carrier Protein Examples: Glucose or amino acids moving from blood into a cell. An nerve electrical impulse results from opening protein channels for ions that move by facilitated diffusion. Factors which affect the rate of Passive Transport Temperature – With increase in temperature average speed and kinetic energy of the molecule increases. faster the molecules move, the faster they diffuse… the slower the molecules move, the slower they diffuse. Pressure – as you increase or decrease pressure…. You can affect the rate and direction of ﬂow. Concentration – the larger the population of solutes, the greater the chance of random access through a membrane. Passive Transport Solutes ﬂow down the concentration gradient * The cell does not use any energy The 3 most common types of Passive Transport are: 1. Diffusion 2. Osmosis 3. Facilitated Diffusion Passive Transport cell uses no energy molecules move randomly Molecules spread out from an area of high concentration to an area of low concentration. (High Low) Three types: Passive Transport 1. Diffusion 1. Diffusion: random movement of particles from an area of high concentration to an area of low concentration. Diffusion continues until all molecules are evenly spaced (equilibrium is reached) Note: molecules will still move around but stay spread out. Passive Transport: 2. Osmosis Osmosis: diffusion of water through a selectively permeable membrane Water moves from high water concentration to low concentrations Water moves freely through pores. Solute (green) to large to move across. Osmotic Pressure Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward ﬂow of its pure solvent across a semipermeable membrane. If there are solute molecules only in one side of the system, then the pressure that stops the ﬂow of the solutes is called the osmotic pressure. Turgor Pressure Internal pressure applied to a cell wall when water moves by osmosis out of the cell. The pressure pushes the plasma membrane against the cell wall. Plasmolysis is the loss of Turgor Pressure… therefore the cell collapses. All living cells must be surrounded by Water. These water environments are classiﬁed as by the concentration of solutes in the solution. The environments are classiﬁed as: 1. Isotonic 2. Hypertonic 3. Hypotonic What type of solution are these cells in? A B C Hypertonic Isotonic Hypotonic Dealing osmatic pressure by organisms Bacteria and plants have cell walls that prevent them from over-expanding. In plants the pressure exerted on the cell wall is called tugor pressure. A protist like paramecium has contractile vacuoles that collect water ﬂowing in and pump it out to prevent them from over-expanding. Salt water ﬁsh pump salt out of their specialized gills so they do not dehydrate. Kidneys keep the blood isotonic by remove excess salt and water. Passive Transport: 3. Facilitated Diffusion A B Facilitated diffusion: diffusion of speciﬁc particles through transport proteins found in the membrane Facilitated Diffusion a. Transport Proteins are diffusion (Lipid speciﬁc – they “select” (Channel Bilayer) only certain molecules Protein) to cross the membrane b. Transports larger or charged molecules Carrier Protein Passive Transport: Facilitated Diffusion Glucose Cellular Transport From a molecules High High Concentration Cell Membrane Protein Low Concentration Low channel Transport Through a Protein Go to Section: Examples: Glucose or amino acids moving from blood into a cell. An nerve electrical impulse results from opening protein channels for ions that move by facilitated diffusion. Factors which affect the rate of Passive Transport Temperature – With increase in temperature average speed and kinetic energy of the molecule increases. faster the molecules move, the faster they diffuse… the slower the molecules move, the slower they diffuse. Pressure – as you increase or decrease pressure…. You can affect the rate and direction of ﬂow. Concentration – the larger the population of solutes, the greater the chance of random access through a membrane. Active Transport cell uses energy actively moves molecules to where they are needed Movement from an area of low concentration to an area of high concentration (Low to High) Solutes ﬂow against the concentration gradient. The cell uses energy….ATP. Requires Transport Proteins Active transport Active transport is the movement of molecules across a cell membrane from a region of their lower concentration to a region of their higher concentration in the direction against some gradient or other obstructing factor (often a concentration gradient). Unlike passive transport, which uses the kinetic energy and natural entropy of molecules moving down a gradient, active transport uses cellular energy to move them against a gradient, polar repulsion, or other resistance. Active transport is usually associated with accumulating high concentrations of molecules that the cell needs, such as ions, glucose and amino acids. Primary active transport: If the process uses chemical energy, such as from adenosine triphosphate (ATP), it is termed primary active transport. Secondary active transport involves the use of an electrochemical gradient. Examples of active transport include the uptake of glucose in the intestines in humans and the uptake of mineral ions into root hair cells of plants. Secondary active transporter proteins Secondary active transporter proteins move two molecules at the same time: one against a gradient and the other with its gradient. They are distinguished according to the directionality of the two molecules: antiporter: (also called exchanger or counter-transporter) move a molecule against its gradient and at the same time displaces one or more ions along its gradient. The molecules move in opposite directions. Symporter : move a molecule against its gradient while displacing one or more different ions along their gradient. The molecules move in the same direction. Both can be referred to as co-transporters. Uniport, symport, and antiport of molecules through membranes Uniport Facilitated diffusion carriers mediate transport of a single solute symport A type of cotransport in which a membrane protein (symporter) transports two different molecules or ions across a cell membrane in the same direction. antiport A type of cotransport in which a membrane protein (antiporter) transports two different molecules or ions across a cell membrane in opposite directions. Types of Active TransportSodium Potassium Pumps (Active Transport using proteins) 1. Protein Pumps -transport proteins that require energy to do work Example: Sodium/ Potassium Pumps Protein changes are important in shape to move nerve responses. molecules: this requires energy! Types of Active Transport 2. Endocytosis: taking bulky material into a cell Uses energy Cell membrane in-folds around food particle “cell eating” forms food vacuole & digests food This is how white blood cells eat bacteria! Types of Active Transport 3. Exocytosis: Forces material out of cell in bulk membrane surrounding the material fuses with cell membrane Cell changes shape – requires energy EX: Hormones or wastes released from cell Models of Pumps P-type ATPase pumps (primary transporters): + + Na -K 2pump 2+ Ca pump Co-transporters: + Na -glucose pump + 2+ Na - Ca exchange Pumps A pump is a protein that hydrolyses ATP in order to transport a particular solute through a membrane in order to generate an electrochemical gradient to confer certain membrane potential characteristics on it. Sodium Potassium Pump showing alpha and beta units + + Na /K -ATPase was discovered by Jens Christian Skou in 1957 while working as assistant professor at the University of Aarhus, Denmark. In 1997, he received one-half of the Nobel Prize in Chemistry "for the ﬁrst discovery of an ion- + transporting enzyme, Na + , K -ATPase. SODIUM – POTASSIUM PUMP Animation Sodium Potassium Pump One of the most important pumps in animal cells is the sodium potassium pump, that operates through the following mechanism: + 1. Binding of three Na ions to their active sites on the pump which are bound to ATP. 2. ATP is hydrolyzed leading to phosphorylation of the cytoplasmic side of the pump, this induces a structure change in the protein. The phosphorylation is caused by the transfer of the terminal phosphate group of ATP to a residue of aspartate in the transport protein and the subsequent release of ADP. + 3. The structure change in the pump exposes the Na to the exterior. The + phosphorylated form of the pump has a low aﬃnity for Na Ions so they are released. SODIUM – POTASSIUM PUMP Transmembrane protein 4 subunits 2 large/ 2 small – 3 Na+ binding sites – 2K+ binding sites – ATPase function – 1 site which is phosphorylated + + 3 Na moved out, 2 K moved in + + 4.Once the Na ions are liberated, the pump binds two molecules of K to their respective binding sites on the extracellular face of the transport protein. This causes the dephosphorylation of the pump, reverting it to + its previous conformational state, transporting the K ions into the cell. + 5.The dephosphorylated form of the pump has a higher aﬃnity for Na + + ions than K ions, so the two bound K ions are released into the cytosol. ATP binds, and the process starts again. SODIUM – POTASSIUM PUMP Works by cyclical process ﬂip-ﬂopping between two conformation Flip-ﬂop governed by phosphorylation 3 Na+ bind on cytoplasmic surface Binding changes conformation activating ATPase ATPase phosphorylates the protein, hydrolysing ATP Causes further conformational change Moves 3Na+ to extracellular side Released because phosphorylation also reduces aﬃnity of protein for Na+ (conformational change) 2 K+ bind on extracellular side (conformational change) Dephosphorylation occurs (conformational change) Restores original conformation Protein ﬂips back to cytoplasmic side taking 2K+ with it 2K+ released The Sodium-Potassium Pump Extracellular ﬂuid with high + concentration Na + + K of Na + Na P + P K Cytoplasm ATP ADP P with high concentration + of K 1 2 3 4 5 + + Three Na bind Phosphate is Phosphorylation K binds to Release of to the transferred changes the the protein, phosphate cytoplasmic from ATP to shape of the causing changes the side of the the protein. protein, moving phosphate shape of the + protein. Na across the release. protein, + membrane. moving K to the cytoplasm. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Calcium Pump Calcium Pumps Calcium is kept at low concentration in the cell by ATP- + + driven calcium pump similar to Na -K pump with the exception that it does not transport a second solute Calcium is tightly regulated as it can inﬂuence many other molecules in the cytoplasm Inﬂux of calcium is usually the trigger of cell signaling 2+ Ca ATPase is aCalcium form ofATPase P-ATPase that transfers calcium after a muscle has contracted. The calcium ATPase are: 2+ Plasma membrane Ca ATPase (PMCA) 2+ Sarcoplasmic/Endoplasmic reticulum Ca ATPase (SERCA) Calcium pumps Calcium pumps are a p-type calcium channel family of ion pump in the cells of humans and other mammals. They are able to transport calcium across their associated cell membrane using energy derived from ATP. 2+ There is a very large transmembrane electrochemical gradient of Ca (a calcium ion) driving the entry of the ion into cells, yet it is very important for cells to maintain low 2+ concentrations of Ca for proper cell signalling; thus it is necessary for the cell to 2+ employ ion pumps to remove the Ca. Failure to do so is one cause of muscle cramping. 2+ The PMCA and SERCA are together the main regulators of intracellular Ca concentrations. Calcium pump structure Mitochondria and lysosomes also serve as reservoirs ++ for Ca subject to release under certain conditions. ++ + Ca -binding proteins in the ER lumen "buffer" free [Ca + ++ ], and increase the capacity for Ca storage. ++ ++ ER Ca -binding proteins have 20-50 low-aﬃnity Ca - binding sites per molecule, consisting of acidic residues. Examples: Calmodulin is a low molecular weight, acidic, calcium binding protein in cytoplasm which mediates the Ca2+ regulation Calsequestrin is in the lumen of the sarcoplasmic reticulum (SR), a specialized ER of muscle. Calreticulin is in the lumen of the ER of non-muscle cells. It also has a role in protein folding. Muscle ﬁber stimulated 2+ Ca released into the cytoplasm from ECF 2+ Ca binds with calmodulin 2+ Ca /Calmodulin activates mysoin kinase Myosin kinase phosphorylates myosin Myosin can now bind with actin 2+ Ca for smooth muscle contraction comes largely from outside the cell MLCK: Myosin light chain kinase Proton pumps Exocytosis (exo = outside, Cyto = cell) moving substances outside the cell Process of vesicles fusing with the plasma membrane and releasing their content to the outside of the cell. Endocytosis (endo = inside, cyto = cell) Capture of substances outside the cell when the plasma membrane merges to engulf it. ***There are three types of endocytosis 1. phagocytosis 2. pinocytosis 3. receptor-mediated Endocytosis Endocytosis functions: Uptake of substances Transport of protein or lipid components Metabolic or division signaling Defense to microorganisms Pinocytosis occurs when dissolved materials enter a cell. The plasma membrane folds inward to form a channel allowing the liquid to enter. The plasma membrane closes off the channel, encircling the liquid inside a vesicle. Oils enter cells through pinocytosis Phagocytosis (phago = to eat, cyto = cell) Phagocytosis occurs when undissolved solids enter a cell. The plasma membrane wraps around the solid material and engulfs it, forming a vesicle. Phagocytic cells, such as white blood cells (monocytes, macrophage, neutrophils attack and engulf bacteria in the manner. Phagocytosis Predominant cells involved in phagocytosis: unicellular cells macrophages osteoblats trophoblasts Uses of phagocytosis uptake of food particles immuneresponses elimination of aged cells (RBC) Receptor-Mediated Endocytosis Occurs when speciﬁc Molecule being ingested Receptor Protein molecules bind to specialized Ligand “receptors” (proteins) in the plasma membrane. The membrane, the receptors, and the speciﬁc molecules, called ligands, fold inward forming vesicles. Hormones target special “target cells” by receptor- mediated endocytosis. Clatharin mediated endocytosis Clathrin-mediated endocytosis (CME) is a vesicular transport event that facilitates the internalization and recycling of receptors engaged in a variety of processes, including signal transduction (G-protein and tyrosine kinase receptors), nutrient uptake and synaptic vesicle reformation. Two classical examples of CME are iron-bound transferrin recycling and the uptake of low- density lipoprotein (LDL). Pathway of LDL insulin or other hormones – in receptor mediated endocytosis Fate of LDL internalized by receptor-mediated endocytosis Summary Molecule Mode of Transportation O2 Diffusion CO2 Diffusion Diffusion H 2O Glucose Osmosis (C6H12O6) Facilitated Diffusion Ions Large Solids (starch, Specialized Transport etc) Phagocytosis Large Liquids (Oils) Pinocytosis Hormones Receptor mediated

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