RER, Golgi, and Secretion

3. RER, GOLGI, and SECRETION Objectives Ultrastructural features Production pathway followed by proteins Cell biological and biochemical evidence of the pathway Mechanisms for protein sorting Post-translational modification of proteins CYTOSOL – is the liquid matrix inside the cell membrane but outside and around the organelles and inclusions Cytoplasm = cytosol + Membranous organelles Non-membranous organelles And Inclusions Examples of secretory cells Pancreatic acinar cell - digestive enzymes plasma cells Examples of secretory cells Con’t Pancreatic acinar cell - digestive enzymes Plasma cell – antibodies plasma cells plasma cells Gal bladder Examples of secretory cells Con’t Pancreatic acinar cell - digestive enzymes Plasma cell – antibodies Chief cells in stomach Neurons Examples of secretory cells Con’t Neutrophils – lysosomes to kill ingested bacteria All cells housekeeping – cell membranes, lysosomes Neutrophils Secretory Cells have Two types of Release 1. Constitutive - consistently releasing without storage granules e.g., Plasma cell plasma cells Method to detect release types Light microscopy Electron microscopy (TEM) = electron opaque Histochemical reaction creates e-opaque deposit using the enzymatic activity of the peroxidase Plasma cells Typical as seen by TEM Dense reaction product is seen in the cisternae of the RER indicating the location of antibody protein in plasma cells with no storage of antibody in secretory granules Evidence for Constitutive secretion of plasma cells horseradish peroxidase reaction product is electron opaque with EM Histochemistry reaction where by native enzymes within the mature face of Golgi reacted with substrate to produce electron opaque participate Histochemistry reaction Nucleus is clear Of rx product Injection of animal with horseradish peroxidase caused its plasma cells to produce antibodies against peroxidase (and the antibodies are located where they are produced in the cisternae of RER) and bind to peroxidase when exposed to the cell by experimental treatment followed by histochemical reaction induction of a electron opaque participate Secretory Cells have Two types of Release 2. Induced – release from storage granules after signal received (hormone/antibody binding) e.g., Pancreatic acinar cell and mast cell Insulin induces release of secretions Pancreatic acinar cell MAST CELLS with granules Receptors on mast cell surface bind IgE antibodies and cAMP is produces inside the cell when the antibodies bind to their specific antigens This allows several antigens to induce mast cell degranulation depending on antigen present. Induced degranulation of mast cells Antibody-antigen induced mechanism makes mast cells respond to specific antigens. Hence, it gives specificity to mast cells. 19709 Mast cells in stained testicular tissue Mast cells Endoplasmic reticulum in secretion General: • Membrane-bound canaliculi • Loose network of branching and anastomosing tubules (SER) Endoplasmic reticulum in secretion General: • Membrane-bound canaliculi • Loose network of branching and anastomosing tubules (SER) • Cisternae - flattened sacks (RER) with ribosomes SER RER Nerve cell body Ribosomes make RER rough • Ribosomal RNA and protein • Basophilic due to phosphate groups – (Nissl bodies of nerve cell bodies) • Polyribosomes - group attached by common mRNA carrying code for amino acid sequence • Function-decoding (translating) mRNA during protein synthesis Nissl bodies Ribosomes on RER started their protein synthesis as free ribosomes and then became attached to RER. Scaler reaction Vectorial reaction Reactions • Scaler reactions a+b=c • Vectorial reactions a+b = Membranes separate c Ribosomes Nissl bodies of nerve cell bodies = ribosomes Nerve cell bodies Ribosomes Free – intracellular proteins Attached to RER – secretory proteins • Signal sequence - attach new protein to RER • Stop transfer sequence • Docking protein on RER for protein synthesis to continue • Ribophorins - attach ribosomes to RER Rough endoplasmic reticulum Ribosomes Protein synthesis and initial glycosylation > Golgi > plasma membrane Nuclear envelope Golgi in secretion • Present in nearly all eukaryotic cells • Function - concentration, chemical modification and packaging of secretory products and production of lysosomes Golgi • Structural and functional polarity • Glycosylation of proteins from RER Cis face Trans face CYTOSOL – is the liquid matrix inside the cell membrane but outside and around the organelles and inclusions cytosol = inactivated protein Structural and functional polarity Cells that use the same pathway of protein secretion • • • • • • • Pancreatic acinar cell Plasma cell Mast cells Neutrophils Chief cells Spermatids Most all cells Key characteristics of the pathway • Directional • Deliver from organelle to organelle by small vesicles • Specificity of end destination Evidence for protein pathway Temporal appearance of radioactive proteins in different organelles (Produced from radioactive precursors e.g., labeled AA) Detected by: Autoradiography visual LM EM Cell fractionation biochemical Evidence for protein pathway Autoradiography Procedure to localize a product (e.g., protein) within a cell or gel that is selfradioactive due to the cell’s incorporation of radioactive precursors (e.g., radioactive amino acids) into that product that is visualized in a photographic emulsion. A pulse – chase experiment to detect the temporal appearance of radioactive proteins in different organelles Pulse = radioactive precursors Chase = non-radioactive precursors following the pulse Do the : Pulse – chase experiment Look for the : Temporal appearance of silver grains in the photographic emulsion over organelles of interest Temporal differences = changes with time Temporal appearance = sequence / timing of occurrence When compared to a cell: B = RER, B/C = Golgi, C/D = Secretory granules, and D = Secretory products Pulse followed with a chase Pulse followed with continued pulse (i.e., no chase) only Results are more difficult to follow (interpret) Note: Good science uses different methods to answer the same question. Cell fractionation Grind cell into components, separate components on sucrose gradient by ultra centrifugation, and analyze components for radioactivity. Separate smooth (Golgi) and rough (RER) vesicles smooth rough Temporal appearance of radioactive proteins in different organelles with smooth or rough membranes Golgi RER Seen by: cell fractionation Temporal differences = changes with time Temporal appearance of radioactive proteins in different organelles with smooth or rough membranes Golgi RER Seen by: cell fractionation autoradiography rough (RER) vesicles Temporal differences = changes with time smooth (Golgi) vesicles Protein sorting? 1. How does a cell segregate cytoplasmic proteins from those for secretion? 2. What signals direct vesicles to a specific organelle? 3. How are proteins designated to be inserted into the membrane or not? 4. How do proteins loop in the membrane? Cell membrane Secretory vesicle Lysosome vesicle Protein No sugar on this side of these membrane proteins Sugars are on membrane proteins out side of the cell but not on proteins inside of the cell. Membrane proteins inside of the cell were never exposed to Golgi enzymes that add sugars. Protein sorting In vitro vs. In situ translation systems for protein production (in vitro translation yielded proteins with 20 hydrophobic AAs more) 1. 2. 3. 4. 5. Signal peptide (~20 hydrophobic AAs) Signal recognition particle Ribophorin - on RER, binds ribosome to RER Stop transfer sequence Hydrophobic amino acid sequence – inserted that portion of the protein into middle of membrane (lipid bilayer) Signal recognition particle Signal peptide (~20 AAs) Ribophorin - on RER, binds ribosome to RER Free ribosomes RER bound ribosomes Signal peptide (~20 hydrophobic AAs) gets stuck in the hydrophobic portion of the mRER membrane Signal peptide (~20 hydrophobic AAs) gets stuck in the hydrophobic portion of the mRER membrane Both = ~20 hydrophobic AAs Location of amino terminus Protein sorting summary 1. If Signal peptide, it goes to RER and start transfer 2. Signal recognition particle 3. Ribophorin on RER, binds ribosome and attaches ribosome to RER 4. Stop transfer 5. Hydrophobic amino acid sequence – inserted into membrane Another option is for the protein to stay in the membrane and become a cell membrane protein For secretion For membrane Membranes are made in the endoplasmic reticulum Membranes Post-translational modifications RER – Initial glycosylation & trim glucose – Add chains of sugars first synthesized on a dolichol [lipid linked (through phosphate groups) oligosaccharide] Post-translational modifications Golgi – Trim glucose & some mannose added by RER Golgi – polarized shape and function Cis • Convex region - phosphate groups added Middle • Mannose removed Trans • Concave region – sialic acid, galactose added Cis face Golgi Cytosol Transport vesicles Black acid phosphatase precipitates in the developing acrosome Trans face Nucleus Spermatid showing the developing acrosome over its nucleus. Acid phosphatase enzymes (black precipitates) first appear in the trans face of the Golgi apparatus and are transferred to the developing acrosome via transport vesicles. Golgi Testis Acrosomic vesicle of a spermatid Golgi - polarized shape and function • Cis – Convex region - phosphate groups added • Middle – Mannose removed – N-acetylglucosamine • Trans – Concave region – sialic acid, galactose added Golgi Also adds fatty acids, sulfate groups Recycling of membrane of transport vesicles from Golgi to RER; however, the cargo does not recycle Golgi and plasma membrane proteins Cell membrane proteins are gylcolasolated only on the out side as that is the only part of the protein that had access to Golgi enzymes that add sugars https://en.wikipedia.org/ wiki/Golgi_apparatus Cell membrane protein Lysosomal pathway • Mannose-6-PO4 directs vesicles with polypeptide to lysosomes Cisternal and postcisternal phases of secretion? Brefeldin A inhibits transport of proteins from ER to Golgi Golgi recycling membrane to RER Nocodazole acts on cells by interfering with the polymerization of microtubules. Specialized cell junctions (tight junctions) isolate the membrane fluidity to luminal or basal/lateral domains of the cell membrane Scaler rx Vectorial rx Signal Peptide on newly synthesizes protein being produced directs free ribosome to RER Scaler rx Vectorial rx Peroxisomes 1. O2 metabolism 2. Detoxification of harmful substances in fatty acid metabolism 3. Crystalline core Next time, but do not go yet lesson on taking the quizzes Lysosomes, smooth ER, mitochondria, and inclusions Approach for studying for quizzzes part A basic tissues Epithelium Connective tissue Protein production / secretion – structure/process Ribosome Ribophorin Dolichol Post-translational modification Protein sorting classes of cellular structures Non-membranous organelles Membranous organelles Approach for studying for quizs part A con’t Characteristic of membranes • Cellular compartmentalization Chemical heterogeneity of the cell Amphipathic molecules Organelles Nuclear envelope Peroxisome Microsopy Resolution Procedures in cell biology Autoradiography Cell fractionation SDS gel electrophoresis Part B 8. Based on the following characteristics, name the basic type of tissue and give one function of the stated characteristic: Characteristic Tissue Type Function a. Gap junction Epithelium Allows cell-to-cell communication b. Avascular Epithelium Blood vessels don’t interfere in function c. Histologic glue Connective tissue d. Myelin sheath Nervous tissue Attach tissues Aid in impulse conduction Part B con’t 9. List the corresponding cellular organelle and the function of the following structures or distinct structural characteristics. Characteristic Organelle Function a. Phospholipid bilayer Membrane Compartmentalization b. Functions in decoding Ribosomes c. Signal proteolysis RER d. Phosphorylation & sulfation Golgi Produce proteins Separate signal from secretory protein Post translational modifications of proteins Part B con’t 10 Contrast light (bright field) with conventional transmission electron (TEM) or scanning electron (SEM) microscopy. Microscope Type of Observation a. Light b. TEM c. SEM Light Source Lens Type Method Part C 13. What is a distinguishing feature of this cell? (Neutrophil) Granular, light staining cytoplasm, lobulated nucleus. 14. What would eosin stain in these cells? (Apical cytoplasm pancreatic acinar cells) secretory granules, protein in cytoplasm. 15. Name two functions of this organelle. (Mitochondrium) (a) ATP production; (b) Ca++ storage; or cholesterol-side chain cleavage. 16. Which is greatest? The thickness of a plasma membrane (7-10 nm), thickness of a microtubule (25 nm), or diameter of a ribosome (15 nm)? Microtubule