Introduction of Cell, Cell Theory and History Chapter 2 Part 2

▪ ▪ ▪ ▪ ▪ ▪ ▪ Membrane Domains/ specialized regions: The regions or domains where only specific proteins are present on the membrane of cell or organelle. EXAMPLE: Epithelial cell of gut – uptake nutrients from the gut by proteins – confined to the apical surface of cell (surface that faces the gut) & proteins involved in export of solutes out – confined to the basal and lateral surfaces. – Barriers are developed when the sides of cells sealed with the other cell from lateral sides by TIGHT JUNCTIONS. Cell Surface – Carbohydrates Majority of proteins on the surface of cell – attached with short oligosaccharides – glycoproteins Certain polypeptides attached with polysaccharides – peptidoglycans In all cases – carbohydrates attached on the outer surface of cell – forms a sugar coating – glycocalyx ▪ ▪ ▪ ▪ ▪ ▪ Functions: Provide protection Oligo and polysaccharides absorb water which make the cell surface slimy Help the cells to change shape by lubricating cells surface Cellular signaling ▪ CHAPTER: 08 (CYTOPLASMIC MEMBRANE SYSTEM; STURCUTRE AND FUNCTION) Development of Electron microscope – 1940s – membrane bounded structures Early micorscopists – saw membrane bounded vesicles, long channels bounded by membranes which form an interconnected network of canals and stack of membrane bounded sacs called cisternae. Eukaryotic cell – occupied by membranous structures Each of these organelles contain a particular compartment of proteins and involved in a particular function. Compartmentalization – division of functions in different locations. Endomembranous system: ER, Golgi apparatus, Endosomes, Lysosomes, Vacuoles – each unit work function in coordination with other components. ** A dynamic, integrated network in which materials are shuttled back and forth from one part of the cell to another part of cell. ** Transportation is carried out in vesicles called transport vesicles – move via cytoplasm in a directed manner – operated by tracks formed by microtubules and microfilaments of cytoskeleton. At destination, these vesicle fuse with the acceptor compartment which receive the cargo along with its membranous wrapper. Biosynthetic pathway/ Secretory pathway: Protein synthesis → ER → Modification → Golgi apparatus → Golgi or transport vesicles → transport them to the destination → plasma membrane, lysosomes, vacuole. *** Most of the proteins and polysaccharides which are synthesized by ER are secreted outside the cell. Secretory activities of cell: Constitutive secretion: Materials → Secretory vesicles → Take from site of synthesis → Discharge into extracellular space with continual manner. Help in formation of extracellular matrix Help in formation of plasma membrane Regulated secretion: Materials → Stored in membrane bounded vesicles → Stimulus → Discharge Large, densely packed membrane-bounded vesicles called secretory granules. Example: Endocrine cells release hormones Synthesis and transport of proteins: Classes of proteins: 1) In-Soluble proteins: discharge out of cell 2) Integral proteins: attached to various membranes 3) Soluble proteins: reside with in compartment of cell **Secretory pathway: Material move out of the cell **Endocytic pathway: Material move within the cell Traffic patterns: for the appropriate delivery of materials to the target location. EXAMPLE: Salivary glands → Proteins synthesized on ER → Targeted to secretory granule While lysosomal enzymes are specifically targeted to lysosomes after synthesis on ER. Transport of proteins to their target location require a specific address or “sorting signal”. Sorting signal: small stretch of amino acids or oligosaccharides – recognized by specific receptors that reside in the membranes or surface coats of budding vesicles – ensure transport to the target location. *Proteins which are the part of complex distribution system consist of soluble proteins that are recruited to specific membrane surfaces. ***Motor Proteins and cytoskeletal elements → Help in transport of vesicles Study of endomembrane: Insights Gained from Autoradiography The cells with high secretory activity have high or abundant amount of ER. Pancreas _ acinar cells_ Secrete abundant enzymes _ have abundant ER How proteins are synthesized and transported to the surface of cell? This could be visualized by autoradiography _ George Palade and James Jamieson Principle: the technique to visualize or determine the location of radioactively labeled materials with the cell. Molecules are covered with photographic emulsion _ exposed to radiations _ revealed under microscope. Experiment: Palade and Jamieson – incubated slices of pancreatic tissue in a solution containing radioactive amino acids for a brief period. Amino acids _ taken up by _ incorporation in to digestive enzymes – Synthesized by ribosomes – location of radioactively labeled proteins determined by autoradiograph – ER the site of synthesis. Second Experiment (Pulse chase experiment): Incubation of tissue in radioactive amino acids – pulse refer to the brief incubation with radioactive amino acids incorporated in proteins – chase refer to the period during which additional additional proteins are synthesized using non-radioactive material – longer time – farther the radioactive proteins manufactured during the pulse will have traveled from their site of synthesis within the cell. Longer the chase, the farther the radioactive proteins manufactured during the pulse will have traveled from the site of synthesis with in the cell. – by this we can easily trace the movement and location of proteins. These experiments also help to tie number of seemingly different membranous compartments into an integral functional unit. Insights Gained from the Use of the Green Fluorescent Protein Alternative technique – utilizes a gene isolated from a jellyfish that encodes a small protein called green fluorescent protein (GFP) which emits green fluorescent light. Technique – Gene which encode GFP is attached with the gene encoding your protein of interest (Chimeric DNA) – Inside the cell – Expression – Chimeric protein consisting of GFP fused at the end of protein to be studied (has no or little effect on protein function and movement). Example: Cell – infected with vesicular stomatitis virus (VSV) – Viral gene (VSVG) fused with GFP gene – expression – VSVG protein in ER – traffic through Golgi complex – transported to plasma membrane – incorporated in viral envelope – Precise then earlier method as it provides a synchronized wave of protein movement. Insights Gained from the Biochemical Analysis of Subcellular Fractions Both the above discussed techniques did not provide the chemical composition of these structures. Technique – Homogenization – break cell and separate their organelles – Albert Claude and Christian De Duve. Homogenization – Cell membrane fragmented – membrane fragments form small vesicles <100nm. – vesicles derived from different organelles have different properties – Allow them to separate from one another – Subcellular fractionation. Membranous vesicles from endomembranous system – Heterogenous collection of similar vesicles Microsomes – microsomal fractions – further fractionated into smooth and rough membrane fractions by gradient technique – Chemical properties can be determined. An organelle separated – Its proteins can be extracted, separated and identified by mass spectrometry. Insights Gained from the Use of Cell-Free Systems Isolated parts of cell perform remarkable activities – Cell Free Systems – they did not contain whole cell – provide information – difficult to study in complex environment of cell. George Palade and colleagues – Studied rough microsomal fraction – derived from RER – striped out small particles attached to rough microsomal particles – capable of synthesizing proteins when provided with required ingredients. Proteins synthesized on ribosomes – released in medium of test tube Proteins synthesized by ribosomes on intact rough microsomes – trapped within the lumen of the membranous vesicle – membranes not only involved in attaching amino acids in proteins but they sequester the proteins with in the ER cisternal space. Insights Gained from the Study of Mutant Phenotypes Mutant – Contain 1 or more genes which produce abnormal protein – Deficiency – Provide the insight of importance and function of that protein. Yeast – vesicles bud from ER – travel to Golgi complex and fuse Researchers – mutants – abnormal distribution of membranes – lack proteins – involved in the formation of vesicles at the ER – In the absences of vesicles formation – mutant cell – Accumulate expanded ER. Cell – mutation in gene – encode protein – involved in vesicle fusion – mutant cells – accumulate – unfused vesicles. All these experiments – ER activities are conserved in all type of cells Proteins are also remarkably similar – structural diversity of cells depends upon the underlying molecular similarities. RNA interference (RNAi) technology – Cell produced small RNA (siRNA) – bind to specific mRNA complementarily – inhibit translation – scientists could use this technique to virtually inhibit the translation of any type of protein. Each mRNA describes the expression of a specific gene – by inhibiting the mRNA the effect of gene on a particular process can be studied. Experiment: Cultured Drosophila cells – identification of gene involved in localization of mannosidase II (synthesized in ER and transported Golgi where bind to the residues) Control cell – synthesizing GFP-labeled version of mannosidase II – fluorescence become localized in Golgi complex siRNA molecules – caused a relocation of GFP-mannosidase into ER – previous seen to be fused with golgi complex – This seems to the absence of a particular protein involved in transport form ER to Golgi The Endoplasmic Reticulum ER – comprises of network of membranes – penetrate much of the cytoplasm Evolved from invaginations of plasma membrane Within ER – Extensive space or lumen – separated from external cytosol by ER membrane. Composition of Luminal or cisternal space – different from cytosolic space Dynamic structure – undergo continual turnover and reorganization. Parts: RER: Presence of ribosomes bound to cytosolic surface Contain network of flattened sacs called cisternae Continuous with nuclear membrane SER: No associated ribosomes Highly curved, tubular, forming an interconnected system of pipelines All the proteins share the same proteins, suggesting that they are involved in common activities including synthesis of specific proteins or lipids At the same time numerous proteins are found only in one or the other type of ER Example: Curvatures of SER tubules – induced by presence of membrane bending proteins called Reticulons which are largely absent in the flattened membranes of RER. On the other hand – RER – Proteins – movement of nascent proteins within the ER lumen. Ratio of SER and RER in cell varies depending upon the functionality of cell. Example: Cells – secretions – like pancreas or salivary glands = extensive RER The Smooth Endoplasmic Reticulum Extensively developed in Skeletal, kidney tubules and steroid-producing endocrine cells. FUNCTIONS: 1- Synthesis of steroidal hormones – endocrine cells of gonads and adrenal cortex 2- Detoxification: Liver – wide variety of organic compounds i.e. barbiturates and ethanol – chronic use of these products – proliferation of SER in liver cells -Performed by oxygen-transferring enzymes (Oxygenases) i.e. cytochrome p450 family. – oxidize thousands of different hydrophobic compounds and convert them to hydrophilic – readily excreted derivatives Adverse effect: harmless compound – benzo pyrene -formed when meat is charred on grill – convert into a carcinogenic protein by detoxifying enzymes of SER 3- Sequestering Ca ions – release of Ca from SER of skeletal and cardiac muscles cells trigger the contraction. Functions of the Rough Endoplasmic Reticulum Starting point – biosynthetic pathway – site of synthesis of proteins, carbohydrates chains and phospholipids Synthesis of Proteins on Membrane-Bound versus Free Ribosomes Polypeptides are synthesized at 2 locales with in the cell. 1- 1/3 proteins encoded by mammalian genome – synthesized by ribosome attached to RER – including: a) secretory proteins b) integral proteins c) soluble proteins (resides with in the compartments of ER, Golgi, lysosomes, endosomes, vesicles and plant vacuoles). 2- Free ribosomes – synthesized proteins – released in cytosol – including: a) Proteins distinct to remain in cytoplasm i.e. enzymes for glycolysis and proteins of cytoskeleton b) peripheral proteins of the cytosolic surface of membrane c) proteins which are transported to the nucleus d) proteins that are integrated into the peroxisomes, chloroplasts and mitochondria. Proteins in the latter two groups are synthesized to completion in the cytosol and then imported post-translationally into the appropriate organelle across its boundary membrane. LOCATION OF PROTEIN SYNTHESIS Determined by the sequence present at the N-terminal portion of the polypeptide. 1- Secretory proteins contain single sequence at N-terminus – direct the polypeptide and ribosomes on the ER membrane. 2- Polypeptide moves into the cisternal space of ER via protein-lined aqueous channels This proposal – Signal hypothesis. Synthesis of Secretory, Lysosomal, or Plant Vacuolar Proteins on Membrane-Bound Ribosomes: Synthesis of polypeptide begins – mRNA binds to free ribosome – polypeptide synthesis – emerge from ribosomes – recognized by signal recognition particle (SRP) (7SL RNA – consist of 6 distinct polypeptides and small RNA molecule) – binds to both signal sequence and ribosomes – temporarily arrest the polypeptide synthesis. Bound SRP – Tag – Enables the complex (SRP-Ribosomes-Nascent Polypeptide) – to bind specifically to the cytosolic surface of ER membrane. * polypeptide synthesized on membrane-bound ribosomes contain a signal sequence (stretch of 6-15 hydrophobic amino acid residues – target the nascent polypeptide to the ER membrane and leads to compartmentalization of polypeptide in ER lumen). Binding with ER – 2 sequential interactions 1- SRP and SRP receptor interaction 2- Ribosome and Translocon interaction (** Translocon: Protein-lined channel embedded in ER membrane through which the nascent polypeptide moves in the lumen of ER) Under normal conditions, the translocon channels remain closed to prevent unwanted flow of Ca. Upon binding of SRP-ribosome-nascent polypeptide chain complex to ER membrane – SRP is released from receptor – ribosomes become attached to translocon – opening of translocon channel – growing polypeptide translocated via hydrophobic pore ring into the ER lumen (pore ring – diameter – 5 to 8 angstrom) – membrane bounded ribosomes will release – translocon channel closed. GTP-binding protein (G protein) – key role in many different cellular processes. Conformation of G protein: 1- One containing a bound GTP molecule 2- other with containing bound GDP molecule. – both have different conformations G protein act as molecular switcher - GTP bound protein – turns the process on – Hydrolysis of the bound GTP turns it off. – in the above given process both SRP and SRP receptor are G proteins. Both interact with one another in their GTP-bound states – hydrolysis of GTP – trigger the release of signal sequence by the SRP – insertion into the translocon. Processing of Newly Synthesized Proteins in the Endoplasmic Reticulum Enzymatic treatment of nascent polypeptide 1- N-terminal portion containing signal peptide – Removed by proteolytic enzymes (signal peptidase) – addition of the nascent protein – by olicosaccharyltransferase Both these enzymes are the integral proteins of ER membrane which target the nascent polypeptide as it enters the ER lumen. 2- RER lumen – Molecular Cheprones – Bind to unfolded or misfolded protein – correct or native 3D conformation RER lumen – protein disulfide isomerase (PDI) – convert -SH groups into -S-S- bonds (disulfide bond) – maintain stability of proteins. Synthesis of Integral Membrane Proteins on Membrane-Bound Ribosomes Integral proteins – RER ribosomes – Translocation – within lumen of ER Integral Proteins – have hydrophobic transmembrane segments – shunted from channel of translocon into the lipid bilayer. X-ray crystallography of translocon – clam-shaped conformation – Channel is open from one side – lateral gate – continually opens and close – provide the opportunity to pass into the lateral lipid layer. **nascent polypeptides – sufficiently hydrophobic – dissolve in lipid bilayer – become transmembrane protein. More hydrophobic protein – more likelihood to pass through the wall of the translocon and become integral protein. Alignment of protein in membrane – presence of positively charged amino acids – translocon – orient – more positive part – towards the cytosol ****For these proteins, their arrangement within the membrane is determined by the direction in which the first transmembrane segment is inserted. Once that has been determined, every other transmembrane segment has to be rotated 180 degree before it can exit the translocon. Membrane Biosynthesis in the ER Membrane – grow – incorporation of newly synthesized proteins and lipids ER lumen – High Ca concentration, abundant proteins with disulfide bonds and carbs chains – similar to extracellular matrix. Synthesis of membrane lipids: Membrane lipids – Synthesized in ER Exceptions: 1- Sphingomyelin and Glycolipids – In ER 2- Some unique lipids of mitochondria and chloroplast lipids are synthesized in their membranes – Have enzymes facing towards the cytosol. Lipids from ER to Golgi and to plasma membrane are carried out in the form of components in membrane of transport vesicles. Membranes of different organelles contain various types of the lipids: 1- Enzymes converting one type of lipids to another type: Example: phosphatidylserine to phosphatidylcholine 2- various vesicle bud off from compartment – one type of lipids come in vesicle while others not 3- Lipid-transfer proteins: transport lipids via aqueous cytosol GLYCOSYLATION IN RER All the proteins synthesized on membrane bound ribosomes becomes glycoproteins. Role of Carbs: 1- binding site as they interact with other macromolecules 2- help in proper folding and stabilization of proteins Glycosyltransferases – bound oligosaccharides to the proteins Each enzyme transfers a specific monosaccharide from a nucleotide sugar i.e. GDP-mannose or UDPN-acetylglucosamine to the growing end of carbohydrate chain. Mostly, oligosaccharides residues attach with asparagine residue of polypeptide residue. Process of Modification: Dolichol phosphate – embedded in ER membrane – sugar added by membrane glycosyltransferases – begins with the transfer of N-acetylglucosamine 1-phosphate followed by N-acetylglucosamine and then 9 mannose and 3 glucose units will be added – Olicosaccharyltransferase – transfer the 14-sugar block to the asparagine residues of polypeptides. Mechanisms that Ensure the Destruction of Misfolded Proteins Misfolded proteins are transported to cytosol by a process called dislocation. In cytosol – oligosaccharides – removed – misfolded proteins are destroyed in proteasomes – Process is called ER-associated degradation (ERAD). Example: cystic fibrosis – mutated cystic fibrosis gene – abnormal protein – destroyed by ER – fails to reach the cell surface. More production of misfolded proteins – accumulation in ER – trigger plan of action – unfolded protein response (UPR). Accumulation of misfolded proteins – activate BiP (a chaperones) in lumen The Golgi Complex Morphology: consisting of primarily of flattened, disklike, membranous cisternae with dilated rims and associated vesicles and tubules. Diameter of cisternae = 0.5 to 1.0 um → arranged in an orderly stack 1 golgi stack → 8 cisternae 1 cell → fewer to several thousand distinct stacks → depending on type and function. Vesicles bud off from a peripheral tubular domain of each cisterna. Golgi complex → flattened cisternae → divided into cis, medial and trans cisternae. Cis / entry face → closest to ER → interconnected network of tubules → cis Golgi network (CGN) → Sorting station → distinguish between proteins which are to be shipped back to ER or those which are allowed to the next Golgi station. Trans / exit face → closest to opposite end of stack → network of tubules and vesicles → trans Golgi network (TGN). → sorting station - -> proteins are segregated into different type of vesicles - -> heading either to the plasma membrane or to various intracellular destination. The membranous elements of the Golgi complex are supported mechanically by peripheral membrane skeleton or scaffold composed of a variety of proteins including members of the spectrin, ankyrin and actin families. A separate group of fibrous proteins are thought to form a Golgi “matrix,” that plays a key role in the disassembly and reassembly of the Golgi complex during mitosis. Difference in composition of the membrane compartments from cis to the trans face reflect the fact that Golgi complex is a processing plant. Newly synthesized proteins/ secretory and lysosomal proteins – leave the ER - Enter Golgi at cis face – pass towards the trans face Glycosylation in the Golgi Complex Key player in assembly of carbohydrate components of glycoproteins and glycolipids. As the ER glycoproteins pass through the Golgi, its mannose residues will be removed from the core oligosaccharides and other sugars are added by glycosyltransferases. The sequence in which the sugars are incorporated into oligosaccharides is determined by spatial arrangement of the specific glycosyltransferases. Sialyltrasferase – localized in trans face of the Golgi stack - place sialic acid – terminus of chain Glycosylation carried out in Golgi is quit variable as the glycosylation carried out in ER. Golgi complex is also the site of synthesis of complex polysaccharides including glycosamino-glycan chains of the proteoglycan and pectins and hemicellulose. The Movement of Materials through the Golgi Complex Golgi formed by the fusion of vesicles at cis face which arise from ER. Each cisterna physically moved from the cis to the trans end of the stack – this is known as cisternal maturation model. Vesicular transport model – cargo (Secretory, lysosomes and membrane proteins) – shuttled through Golgi stack – CGN to TGN LYSOSOMES Digestive organelles – animals – 50 different hydrolytic enzymes produced in ER All lysosomal enzymes – work at acidic pH (4.6) – acid hydrolases The high internal concentration is maintained by a proton pump present in membrane of organelle’s boundary. Lysosomal membrane – contain – variety of glycosylated integral proteins – whose carb chains perform protective lining that shields the membrane from the enzyme attack. Role: breakdown of materials brought into the cell from the extracellular environment. Cell – ingest food particle – disassembled by lysosomal enzymes – resulting nutrients – pass via lysosomal membrane into cytosol Phagocytic cells i.e. macrophages and neutrophils – scavengers – ingest debris and dangerous microbes. Ingested bacteria – inactivated by low pH of lysosomes – then enzymatic digestion Digested peptides also posted on cell surface where they alert the immune system. AUTOPHAGY Organelle turnover Regulated destruction of cell’s own organelles and their replacement. Example: Mitochondria – surrounded by double membrane structure (called phagophore) – double-membrane sequestering vesicle – called autophagosome. → outer membrane fuses with lysosome – autolysosome – in which both the inner membrane of autophagosome and enclosed contents are degraded – products will be available to the cell. An estimation – 1 to 1.5% proteins of health liver cells – degraded via autophagy – for cellular renovation. Autophagy – evolved in early eukaryotic organism as a response to nutrient deprivation. Plant/ animal or yeast cell – starvation conditions – increased autophagy – increased cannibalizing their own organelle. Mammalian embryo – before implanted in uterus – for nutrients before it takes from mother’s bloodstream. Atg genes – required for autophagic pathway – deletion – have serious consequences on embryonic development. Provide protection against intracellular threats – like aggregation of abnormal proteins or invading microbes Also help in prevention of neurodegeneration. Example: Blockage of autophagy in certain region of brain – experiences massive loss of nerve cells. After completion of autophagy – remaining body – residual body – contents released by exocytosis or organelle may remain in cells as lipofusin granule. These granules are considered as the major characteristic of the aging process. Plant cell vacuole Plant cell – single large vacuole – 90% of cell space Carry out a wide spectrum of essential functions. Storage: Ions, sugars, amino acids, proteins and polysaccharides – store temporarily Toxic compounds – cyanide (glycoside and glucosinolates) – released when injured by herbivore or fungus. Membrane – tonoplast – contain active transport systems – pump ions into vacuole compartment (much concentrated then normal cytoplasm) – water enters in vacuole by osmosis – hydrostatic pressure – mechanical support and stretches the cell wall during cell growth. Intracellular digestion – not like lysosomes – have some acid hydrolases – low pH is maintained by a V-type HATPase within tonoplast – pumps protons into the vacuolar fluid Proteins of vacuoles are synthesized by ER ribosomes. ENDOCYTOSIS Division: Bulk phase endocytosis and receptors-mediated endocytosis Bulk phase endocytosis/pinocytsis: non-specific uptake of extracellular fluid EXAMPLE: large or small molecules – present in the enclosed fluid – gain entry into the cell Can be visualized – by adding substance to the culture medium – dye lucifer yellow or the enzyme horseradish peroxidase – take up by cells non-specifically. It also helps in the recycling of membrane between the cell-surface and interior compartment. Receptor-mediated endocytosis (RME) – for specific extracellular macromolecules (Ligands) following their binding to receptors ono the external surface of membrane.