Cell Membrane, ER, Ribosomes PDF

Plasma membrane, ER, Ribosomes • Organelles are "little organs" in the cytoplasm Cell membrane (Plasmalemma) • • • • Consists of: o Lipids (mostly phospholipids, e.g. cholesterol) o Proteins (mostly glycoproteins Membrane Lipids. Two parts: one hydrophobic or nonpolar part consisting of fatty aci...

Plasma membrane, ER, Ribosomes • Organelles are "little organs" in the cytoplasm Cell membrane (Plasmalemma) • • • • Consists of: o Lipids (mostly phospholipids, e.g. cholesterol) o Proteins (mostly glycoproteins Membrane Lipids. Two parts: one hydrophobic or nonpolar part consisting of fatty acids; polar or hydrophilic part consisting of Glycerol, Phosphate & Choline. This constituues the hydrophilic head & hydrophobic tail. Together it is an amphipathic molecule. Cholesterol is particulary common, Phosphatidylholine, Sphingomyelin & Glycolipids are quite common, too Two organizations: o Liquid-disordered state - fluid-mosaic model: Phospholipids are loosely packed and capable of rapid lateral diffusion Liquid-ordered state - membrane rafts: microdomains with confined movement of lipids Lipid bilayer is approximately 2 nm broad Membrane Proteins: o Transmembrane: single-pass & multi-pass o Peripheral: ectoperipheral & endoperipheral Cell coat - glycocalyx o Carbohydrate-rich zone on the cell surface, composed of: glycoproteins, glycolipids, proteoglycans o Typically less than 15 oligosaccharides: branched (hundreds of combinations), linked by a variety of bonds, with geat diversity o • • • • Simple diffusion vs. carrier proteins (by change of form) using energy & channel proteins • Functions of membrane proteins: o Structural support o Transfer of signals: • receptors/receptozymes • cell adhesion molecules (CAM) - integrins, selectins, etc. Transport of large molecules: • Endocytosis • Exocytosis o Transport of small molecules • Ion channels: Na, Ca, K, etc. • Ion pumps: Na/K, H/K, Na/Ca, etc. • ABC transporters: P-glycoprotein membrane proves • Aquaporins 0-9 • Glucose transporters • Neurotransmitter transporters o Source of inflammatory lipid mediators Structural Support: o rist• i stural profe found in ayroskeleton ofIn a & • • Spectrin molecule: two chains mingled to a stable structure, with 100 nm pretty large Spectrin dimers interact with specific proteins (junctional complexes) • ASA links: Ankyrin, Spectrin, Actin that link Ankynn's- process to the membrane prorems agloskeleron > oforms a meshwork Spectronwith acn and owner - globular provers long filaments Acrin M > - Glycoprotes:manumgsvaby • protein and we transporting Band Three layers: o Transmembrane: Glycophorins, Band 3 Ankynn Lukstransmembraneng cyroskeleron o Endoperipheral: Ankyrin, Adducin, Band 4.1 Adducin regulates spechn-achi Interaction-stability o Intracellular: Spectrin, Actin Band hilsenacIn a s Clinical correlates of the RBC (Red blood cells) Cytoskeleton: 3 com : : - • sphencal shaped RBC epprical shaped RBCS oval shaped RBCs cannor • are andity anne Signal transduction: Conversion of ectracellular signals into intracellular ones Specu - forms meshwork filaments Achin polymences filament and with form achin • Signal Transduction may take place via lipid rafts (microdomains with confined movement of lipids) • Transport vesicles: Clathrin-coated & Caveolae o Caveolae: Membrane invaginations (50-100 nm) • Consists of lipid rafts and speific proteins: caveolins • Different form: grape-like, rosette, tubular, vesicular • • • • Vesicular transport: ▪ Transcytosis: endolethial cells ▪ Endocytosis: viruses, fungi, bacteria, parasites, prion Cholesterol homeostasis: HDL metabolism Signal transduction Diseases: ▪ Tumorigenesis: caveolin-1 (target of oncogenes and tumor supressor) ▪ Diabetes: caveolae are highly enriched in adipocytes; glucose transporters are associated with caveolae; insulin & leptin signaling involves caveolae ▪ Vascular abnormalities • Cardiomyopathy • Increased vascular relaxation due to increased NO • Impaired angiogenesis o • Atherogenesis: loss of caveolin-1 is protective Clathrin-coated vesicles Transport via transmembrane channels • Aquaporins: different aquaporins do different tramsmission in different organelles • Glucose Transporters (GLUT): • ABC Transporters (ATP-Binding Cassette transporters): A family of 12 or 17 TM proteins • Function: • Unidirectional translocation across membranes • Chemically diverse substrates • Classification: • 7 subfamilies (ABCA – ABCG) • At least 48 members • Ubiquitous expression P(ermeability)-glycoprotein: • 12 TM domains • 170 kDa • Contributes to 90% of cancer deaths (500,000/year in USA) • Exports a variety of anticancer drugs • • Clinical correlates: • Formation and maturation of cell membrane proteins: synthesized in the rough endoplasm reticulum, modified and completed in the Golgi apparatus, transported in vesicles to the cell surface Intracellular membranes ER • • • • • Endoplasmic Reticulum (ER): present in all eukaryotic cells. Consists of rough (coated with ribosomes) and smooth (no ribosomes) ER. The ER is constantly being reorganized o ER membrane ≥ 50% total cellular membrane o ER lumen ≤ 10% of the volume of the cell The ER network is continually reorganizing with some connections being broken while new ones are formed. Motor proteins moving along microtubules pull out sections of ER membranes to form extended tubules that then fuse to form a network. RER: synthesis of transmembrane proteins for ER, plasmalemma, and the membranes of Golgi complex, lysosomes, secretory granules. Glycosylation of proteins to form glycoproteins o Polyribosome-attached membrane enclosed flattened sacs (cisterns) and branching tubules that communicate with each other SER: synthesis of phospholipids and cholesterol for ER, plasmalemma, and the membranes of Golgi complex, lsysosomes & secretory granules; Sequestering Ca2+ Signal Hypothesis (Günter Blobel) • Theory, how a protein identifies its goal: o Signal sequence of polypeptide binds on a Signal Receognition Particle (SRP) o Together they bind to a SRP receptor in the Lumen o The receptor opens a channcel, through that the polypeptide is let in o Finally the protein can be transmitted in the cell • Free polyribosomes (polysomes) o Initial translation of mRNA (up to 70-80 amino-acid containing a sorting signal) o Binding of polypeptide-ribosome complex to SRP • RER o SRP receptor binds polypeptide-ribosome-SRP complex o Polypeptide passage through a RER transmembrane translocator pore into the RER lumen o Signal peptidase cleaves the sorting signal o Elongation of the polypeptide chain o Polypeptide release into the RER lumen or insertion into the RER membrane • The ribosomal cycle: • Signal Hypothesis (cont'd): • Translocation of polypeptides into the RER lumen through a translocator pore in the RER embrane • Cleavage of signal sequence: signal peptidase • Growth (elongation) of polypeptide chains • Posttranslation: RER, Golgi complex • Vesicular transport: RER to cis-Golgi and vice versa • • • Storage in secretory granules, lysosomes or synaptic vesicles, and insertion in the plasmalemma Exocytosis: final steo of secretory pathway Why are polyribosomes bound to the ER, but not to any other organelle? The translocation of polypeptide chain through the pore of the translocator (transmembrane protein complex of RER) occurs usually during translation of mRNA, i. e. co-translationally Note: translocation of proteins into mitochondria and peroxisomes occurs post-translationally, after protein synthesis and release in the cytosol. This explains why polyribosomes are bound to the ER, but not any other organelle. Ribosomes • Function: protein translation Structure: ribosomes re made of rRNA and proteins. • Eukaryotic ribosome structure: • Four rRNAs 28S, 18S, 5.8S, 5S • Plus associated proteins (> 50) • Prokaryotic structure is very similar, but lacks 5.8S rRNA • 4,2 MDa Two subunits: large subunit (60S), small subunit (40S) • • Assembled in nucleus Proteins have to be imported from the cytoplasm into the nucleus • • • • Ribosome function is basically a ribozyme (enzymative) Lots of internal structure through base-pairing Many bases are post-transcriptionally modified Ribosomal Subunits: • The genes responsible for making the rRNAs primary transcript is cut and chemically modified • a large 45S rRNA is copied from the DNA. The source of 3 of 4 rRNAs 28S, 18S, 5.8S • The 5S gene is outside the nucleolus • Ribosome subunit assembly takes place in the nucleolus, bringing together the genes, the 45S primary transcript and the processing enzymes and the proteins. Finished small and large subunits are sent to the cytoplasm separately to do their work: i.e. translation • • Golgi complex, Lysosomes, Peroxisomes, Mitochondria Golgi Apparatus • Parts of the Golgi complex: o Cis Golgi network (CGN) - network of interconnected tubular and cisternal strucures - protein sorting (secretory vs. retrieval proteins) o Collection of 4-6 membrane-enclosed flattened cisternae linked by tubules (dyctiosomes): cis-Golgi, medialGolgi & trans-golgi compartment o Trans Golgi networtk (TGN): network of interconnected tubular and cisternal structures for protein sorting o Golgi matrix proteins involved in the function of the organelle • Functions of Golgi o Sorting (CGN): phosphorylation of oligosaccharides on lysosomal proteins o Cisternae (Golgi stack) Some sugars get removed and replaced by N-acetylneuraminic acid. It is negatively charged and will therefore attract positive charged proteins o Sorting (TGN): Decision where the protein is send to, then release • Two hypotheses explaning the transport in Golgi Apparatus: o Vesicular Transport model: Vesicles move between the cisterns o Cisternal Maturation model: the cisternas themselves move Microtubules serve as a railway whereon the vesicles are moved to the Golgi apparatus • Lysosomes • • • • • Membrane-enclosed structures filled with different types of acid hydrolases (e.g. proteases, nucleases, lipases). The lysosomes limiting membrane and the intralysosomal pH (around 5.0) protect the cytoplasm and other organelles against digestive attack of the lysosomal enzymes (cytoplasmic pH around 7.2) Function: Digestion o Digestion of macromolecules, phagocytosed microorganisms, cellular debris o Digestion of senescent organelles, such as mitochondria and RER o End products are transported into cytosol and are either reused or exportet Acid Hydrolases: o Nucleases o Proteases o Glycosidases o Lipases o Phosphatases o Sulfatases o Sulfatases o Phospholipases The low pH of a Lysosome is achieved by a H+-pump into the cell by using ATP Formation of lysosomes: o Receive their hydrolytic enzymes as well as their membranes from the TGN o Membranes arrive as clathrin-coated vesicles, which fuse with the lysosomal membranes o Hydrolytic enzymes possess mannose-6-phosphate receptors, to which these enzymes are bound. IN the acidic environment of the late endosome, lysosomal enzymes dissociate from their receptors • • • Formation of lysosomes via late endosomes: o Late endosomes contain material received from both the plasma membrane by endocytosis and newly synthesized lysosomal hydrolases, and they therefore already bear a resemblance to lysosomes o There is no real distinction between late endosomes and lysosomes Lysosomes play an important role in the metabolism of several substances in the human body, and consequently many diseases have been ascribed to deficiencies of lysosomal enzymes (lysosomal storage diseases or mucopolysaccharidoses) Lipofuscin: insoluble brownish-yellow graular intracellular material that accumulates as a functon of age or atrophy. Not injurious to the cell but important as a arker of past free-radical injury. Peroxisomes • • • • • • • • • Found in all eukaryotic cells Membrane-enclosed organells Self-replicating Exist without a genome of ist own, must import all proteins Peroxisomes are not only involved in metabolic processes such as hydrogen peroxide detoxification, but also in signaling pathways that promote developmental decisions and cell differentiation in the brain, adipose tissue, placenta, etc. Small (0.2-1.0 micrometers in diameter) sherical to ovoid, membranous organelles Contain more than 40 oxidative enzymes (especially urate oxidase, catalase, and d-amino acid oxidase) Defects of Peroxisomes: Peroxisome Biogenesis Disorders (PBD) or Peroxisome Single Enzyme Disorders (PSED) Functions: • • Biogenesis by peroxisomal targeting signals (PTS1 or PTS2). PTS1 is a tripeptide and is used by more than 90% of human peroxisomal proteins PTS-containing proteins are recognized by PTS cytosolic (soluble) receptors, which belong to the Pex family of proteins. Next steps are: (i) docking of PTS proteins to peroxisomal membrane; (ii) dissociation of the receptorligand complexes; (iii) import of ligand (peroxisomal matrix proteins) into the peroxisome; (iv) recycling of receptors in cytosol. Proteasomes • • • • • • • Not membrane-surrounded Tranlation of mRNA into amino acid sequence is not the end of protein formation. For ist proper function, proteins need to be folded correctly into 3D cnfiguration, and assemble with ist particular subunits chains. Protein folding is mainly done by a proteasome called chaperone Misfolded Proteins are exported from RER and degraded in cytosol by proteasomes The retrotranslocation occurs through the same translocator, through whcih the proteins enter the RER Ubiquitin-Proteasome System (UPS): o Proteasomes are non-membrane-bound organelles consisting of ATP-dependent proteases that form a central cylinder (proteolytic chamber) supplemented by a cap (gate) at each end. o Proteasomes recognize misfolded proteins destined for degradation by their linkage to a small molecule: ubiquitin. Ubiquitin is recognized by a specific receptor in the cap. Misfolded protease-resistant proteins can aggregate causing neurodegenerative disorders o Alzheimer o Huntington o Parkinson o Prion diseases Mitochondira • • • • • Double-membrane-enclosed organelles, most probably evolved from bacteria by endocytosis o Outer membrane, which contains a large number of transport proteins named porins o Inner membrane, which forms many invaginations (cristae) that are rich in enzymes of of the respiratory chain/oxidative phosphorylation. They generate ATP (100 molecules/sec) via the ATP synthase o Intermembrane space o Matrix, which contains genomic apparatus and proteins Functions: o Generation of ATP o Citric acid (Krebs) cycle o Haem biosynthesis o β-oxidation of fatty acids o Apoptosis (programmed cell death) o Calcium storage and signaling o Generation and detoxification of reactive oxygen species ATP hydrolysis (ADP + Pi) drives a large number of ell's energy-requiring processes. Generation of ATP is realized through oxidative phosphorylation, proton gradient in inner membrane, and repeated in changes in ATP synthase's conformation that convert mechanical energy into chemical bond energy. Next, ATP is released from mitochondrial matrix into cytosol to be used, while ADP & Pi enter the matrix for ATP re-synthesis Parts of the mitochondrial complex: o TOM complex (translocator of outer membrane): mediates import of all nucleus-encoded mitochondrial proteins o TIM compllex (translocator of inner membrane): mediates import of all nucleus-encoded mitochondrial proteins o OXA complex: localized in inner membrane, and mediates insertion of inner membrane proteins synthesized in cytosol or mitochondria o SAM complex (sorting and assembly machinery): localized in outer membrane, mediates insertion of outer membrane proteins Transport of nucleus-encoded proteins into mitochondria: o Synthesis on cytosolic polyribosomes o Binding to cytosolic hsp70/hsp90 to keep their unfolded configuration Signal sequence* recognizes import receptor that is component of TOM complex Opening of the pores of TOM and TIM23 at contact sites of inner/outer membranes Cleavage of signal sequence by signal peptidase “Pulling” the protein into matrix* by mitochondrial hsp70 Removal of hsp70 by ATP hydrolysis allowing imported proteins to fold Mitochondria and Apoptosis: Damaged cells commit suicide via apoptosis. This process depends on a family of proteases that have cysteine at heir active site at aspartic acids; hence caspases. In apostosis, mitochondria release the inner membrane protein cytochrome c into the cytosol. Cytochrome c binds/activates an adaptor protein resulting in activation of caspase cascade, which cleaves key cellular proteins leading to apoptosis. Mitochondria also release a protein that blocks apoptosis-inhibiting proteins; this further increases the efficiency of apoptosis o o o o o o • CF = cis face TV = transport vesicles SV = secretory vesicles TF = Trans face G = Golgi apparatus L = Lysosomes Peroxisomes (human) 1. 2. 3. 4. Outer Membrane Lammelar cristae of the inner membrane Tubular cristae of the inner membrane Mitochondrial ribosomes Cytoskeleton • • An intricate cytoplasmic 3D meshwork of protein filaments that are responsible for the cell maintenance, f. e. for: o Cellular morphology o Cellular motion (the entire cell / organells within the cell) Components: o o o Actin-containing filaments (microfilaments): 5-9 nm, composed of actin and actin-associated proteins, twostranded helical polymers; organized into variety of linear bundles, two-dimensional networks & threedimensional gels Microtubules: 25nm, composed of tubulin & microtubule-associated proteins Intermediate filaments: 10nm, composed of homo- or heteropolymers of specific proteins Actin filaments • • • • • • • Actin filaments (also known as microfilaments) are two-stranded helical polymers of the protein actin. They appear as flexible structures, with a diameter of 5-9 nm, and they are organized into a variety of linear bundles, two-dimensional networks, and three-dimensional gels. Although actin filaments are dispersed throughout the cell, they are most highly concentrated in the cortex, just beneath the plasma membrane. Actin filaments are continously assembled and disassembed from G-Actin to F-Actin Type of Actin and their distribution o α - muscle-specific o β - ubiquitous o γ - ubiquitous The two ends of an actin filament polymerize at different rates: o The fast-growing end is called the plus end o The slow-growing end is called the minus-end The difference in the rates of growth is made possible by changes in the conformation of each subunit as it enters the polymer Actin filaments can dynamicly assembly. If a signal comes in, such as a nutrient source, the actin filaments can dissamble and rapidly diffuse into subunits, then reassembly at a new site Glossary: o G-actin: monomeric, globular in configuration (42 kDa) o F-actin: filamentous double-helical assembled G-acting with two ends (+ (growing, barbed) & - (shrinking, pointing)) o o Capping: binding of actin-associated protein to the +-End of F-actin that preents further assembly of Gactin Nucleation: assembly of G-actin subunits into small oligomers o o o • • • • Assembly: non-covalent binding of G-actin to form F-actin Sequestration: binding of G-actin with actin-associated protein Severing: breaking of actin-to-actin-bonds within the filament Actin-associated Proteins: o Actin-sequestering proteins: • Profilin • Thymosin β4 • Gc protein (group-specific component or vit.-D-binding protein) • Gelsolin (brevin) o Actin-severing proteins • Gelsolin • Fragmin • Severin o Actin-crosslinking proteins • Myosin • α-actinin • Dystrophin • Filamin, fimbrin • Villin • ERM proteins (ezrin, radixin, moesin) AF = A + AAP → Actin filaments = Actin + Actin-associated proteins Actin filaments in animal cells are organized into several types of arrays: o Dendritic networks o Bundles o Weblike (gel-like) networks Some actin filament structures are assembled and maintained by two classes of proteins: bundling proteins, which cross-link actin filaments into a parallel array, and gel-forming proteins, which hold two actin filaments together at a large angle to each other, thereby creating a looser meshwork. • Filamin prommotes the formation of a loose and highly viscous geel by clamping together two actin filaments roughly at right angles. • Connections of the cytoskeleton to the plasma membrane: Members of the ERM (named for its first three members, ezrin, radixin, and moesin) help organize membrane domains through their ability to interact with transmembrane proteins and the underlying cytoskeleton. Actin-filament-plasmalemma structures: o Microvilli (brush border): microvillus inclusion disease (Davidson's disease) - inherited disorder of intestinal enterocyte microvilli formation, associated with high mortality and the most common cause of severe refractory diarrhea in the neonate o Lamellipodia and filopodia: cell protusions involved in cell migration o Stereocilia: found in hair cells of the inner ear and epiddidymis o Contractile ring (clevage furrow): involved in cytokinesis - cell division o Cell-to-cell junctions (zonula adherens) o Cell-ECM junctions (focal contacts) Microvilli (sing.: microvillus) are under light microscope microvilli seen as brush border Stereocilia that project from hair cells vibrate in response to sound waves: movement opens stressactivated ion channels in the plasma membrane, leading to membrane depolarization. This is translated into the perception of sound. The human genome includes 40 myosin genes All eukaryotic cells contain myosin, but the richest source of myosin are the muscle cells: o Myosin I: one-headed motor domain o Myosin II: two-headed motor domain o Other myosins are either one- or two-headed • • • • • • Organization of acessory proteins in a sarcomere: Each giant titin molecule extends from the Z disc to the M line. Part of each titin molecule is closely associated with a myosin thick filament; the rest of the titin molecule is elastic and changes length as the sarcomere contracts and relaxes. • • • Actin-specific drugs: o Phalloidin: binds and stabilizes filaments o Cytochalasin: caps filament plus ends A link between AF and ECM is mediated by the protein dystrophin. It connects the extra- and intracellular protectin layer (Glycoprotein complex with Actin cytoskeleton) Dystrophin-associated proteins and human muscular dystrophy (MD): o Dystrophin: Duchenne & Becker MD (DMD, BMD) o Sarcoglycan complex: Limb-girdle MD (LGMD) o Laminin: Congenital MD (CMD) Microtubules • • • • • • • • • Microtubules are structurally more complex than actin filaments, but they are also highly dynamic and play comparably diverse and important roles in the cell. Microtubules are polymers of the protein tubulin. Long, straight, rigid tubular-appearing structures that act as intracellular "railways". With an outer diameter of 25 nm, they are much more rigid than actin filaments Microtubules are long and straight and typcally have one end attached to a single microtubule-organizing center (MTOC) called a centrosome The subunit of each protofilament is a tubulin heterodimer, formed from a tightly linked pair of α- and β-tubulin monomers. Teach protofilament consists of many adjacent subunits with the same orientation. The microtubule is a stiff hollow tube formed from 13 protofilaments aligned in parallel. Microtubules are very dynamic structures; they are continuously assembled and disassembled MT = T + MAP; Microtubules = Tubulin + MT-associated proteins Spatial and temporal organization of molecules and organelles is ensured by their intracellular transport to various destinations within the cytoplasm Glossary: o Tracks (cytoskeletal structures having + and - ends) • Microtubules (MT) • Actin filaments (AF) o Motor proteins: • Kinesins - (+)-end-directed (anterograde) MT motors • Dyneins - (-)-end-directed (retrograde) MT motors • Myosin Vs - (+)end-directed (anterograde) AF motors o Cargo • Membrane-bound organelles • Non-membranous components: mmRNA, IF, viruses • • • • • • • • Human dieseases & motor proteins: o Kinesin gene mutations: • Charcot-Marie-Tooth type 2A disease • Hereditary spastic paraplegia o Myosin V gene mutations: • Griscelli's syndrome: recessive disorder characterized by skin and neurological symptoms o Dynein: Kartagener's syndrom Functions of microtubules: o Intracellular vesicular transport o Positioning of RER & Golgi complex o Structuring of MTOC/centrosome o Structuring of cilia & flagella o Mitotic spindle Microtubule-Organizing Center (MTOC) o While α- and β-tubulin are building units of MT, γ-tubulin is involved in MT nucleation o MT are nucleated from a specific formation known as a MTOC. The centrosome is the major MTOC o The centrosome is composed of 2 centrioles surrounded by amorphous matrix containing γ-tubulin ring complexes (γ-TuRC) that serve as templates nucleating MT growth. o MT are nucleated at their (-)end with the (+)end growing outward from each MTOC Centrosome: o Cylindrical structures, approx. 0.2 μm in diameter and approx. 0.5 μm in length o Each centriole consists of 9 relatively short microtubular triplets linked together in a pinwheel-like arrangement o In the triplets, microtubule A is complete and consists of 13 protofilaments, whereas microtubules B and C share protofilaments o The 2 of centrioles is called centrosome Cellular structures containing MT: o Intracellular: • MTOC/centrosome • Mitotic spindle • Basal body o Plasmalemmal: • Cilium • Primary (immotile) cilium • Flagellum Primary (immotile) cilia: o 9x2 + 0 MTs o IFT proteins o Motor proteins o Key receptors clustered in primary cilia Cilia & flagella (sing.: cilium, flagellum): o Motile processes, covered by cell membrane, with a highly organized microtubule core o Ciliar are normally many, each about 2-3 μm in length o In humans, the spermatozoa are the only cell type with a flagellum Axoneme: core structure of cilia and flagella o 9x2 + 2 microtubules o MTs of the peripheral doublets (9x2) are identified as A (complete with 13 protofilaments) and B (with only 10 protofilaments) o Doublets are linked to each other by protein bridges called nexins o MT motors: dyneins • • • At the base of each cilium or flagellum is a basal body, similar to a centriole, which controls the assembly of the axoneme Ciliopathies (dieseases of cilia or basal bodies): o Joubert syndrome (JBTS) o Bardet-Biedl syndrome (BBS) o Meckel syndrome (MKS) o Ellis Van Ceveld syndrome (EVC) o Oro-facial-digital syndrome type 1 (OFD1) o Jeune syndrome (JATD) Microtubule-specific drugs: o Taxol: binds and stabilizes microtubules o Colchicine / Vinblastine / Nocodazole: binds subunits and prevents their polymerization Intermediate Filaments • • • • • Intermediate filaments are ropelike fibers with a diameter of around 10 nm; they are made of intermediate filament proteins, which constitute a large and heterogenous family. o One type of intermediate filament forms a meshwork called the nuclear lamina just beneath the inner nuclear membrane o Other types etend across the cytoplasm, giving cells mechanical strength. o In epithelial tissue, they span the cytoplasm from one cell-cell junction to another, thereby strengthening the entire epithelium Humans have at least 67 genes that encode IF proteins. This gene family is one of the largest in the human genome. IF are of five types with either cytoplasmic or nuclear location: o Type I-IV - cytoplasmic o Type V - nuclear IF-Functions: o Cellular stability o Cell-to-cell junctions: desmosomes o Cell-tp-ECM junctions: hemidesmosomes o Structuring of nucleus: nuclear lamina The combined surface are of AF, MT and IF exceeds by more than 10 times the area of all cellular membranes. This, together with cross-talk among these cytoskeletal systems, demonstrates the significant potential of the cytoskeleton in the regulation of various cellular functions, • including cell shape and motility, intracellular transport, intercellular and cell-matrix junctions, DNA transcription, RNA processing, cell division, and signal transduction IF Proteins & Human Disease: Summary • • • All eukarytic cells contain actin and tubulin. All eukarytic cells contain actin filaments and microtubules. However, IF (except nuclear lamin-containing IF) are found only in some eukaryotes. Further, most IF proteins are cellselective, i.e. present in only a certain type of cell. Main function of cytoskeletal elements: o Actin filaments: movement o Microtubules: Transport o Intermediate filaments: stability Cytoskeletal component cross-linker Proteins: o AF cross-linkers: • Villin • Fimbrin • Filamin • α-actinin • Spectrin o MT cross-linkers: • MAP1-5 • Tau o IF cross-linkers: • Plectin • filaggrin Cell Junctions • • Several membrane-associated structures contribute to adhesion and communication between ells or between cells and ECM. They are present in most tissues but are particularly numerous and prominent in epithelia Cell Junctions: o Occluding junctions: • Tight junctions (zonula occludens) - keep neigboring cells together in epithelial tissue in a way that prevents even small molecules to pass through the junction --> apical and basolateral surface in gut epithelial cells o Anchoring junctions: • Actin-filament-associated: ▪ Cell-cell junctions (adherens junctions, zonula adherens) ▪ Cell-matrix junctions (focal adhesions) • Intermediate filament-associated ▪ Cell-cell junctions (desmosomes) ▪ Cell-matrix junctions (hemidesmosomes) o Communicating junctions: • • Channel forming cell-cell junctions (nexus, gap junction) Signal relaying cell-cell junctions (chemical synapses) • Tight junctions (TJ): Each tight junction is composed of transmembrane adhesion proteins embedded in each of the two interactin plasma membranes. Extracellular domains of these proteins contact directly to one another, thus occlude the intercellular space o Major TJ proteins: • Claudins: transmembrane proteins • Occludins: transmembrane proteins • ZO proteins - peripheral membrane proteins which associate with actin filaments • Transcellular & paracellular pathways of transepithelial transport: o Transcellular transport requires the cellular uptake of molecules on one side and subsequent release on the opposite side o In paracellular transport, molecules move extracellularly through parts of tight junctions, whose permeability to small molecules and ions depends on the composition of the junctional components and the physiologic state of the epithelial cells Anchoring junctions: o Tramsmembrane adhesion proteins • Cadherins - adhesion junctions & desmosomes • Integrins - focal adhesions & hemidesmosomes o Intracellular anchor proteins - connect the transmembrane proteins with actin or intermediate filaments o Anchoring junctions can also contain components of intracellular signaling Anchoring junctions: adherens junctions o Cell-cell junctions o Form the adhesion belt (zonula adherens) in epithelial cells o Molecular composition of zonula adherens: • Transmembrane adhesion proteins - cadherins • Contractile bundle of AF & myosin just adhacent to the junction • Intracellular anchor proteins - catenins, vinculin, α.actinin o Cadherins mediate cell-cell-adhesion by a homophilic binding Cell-cell-adhesion: o Homophilic binding: molecules on one cell bind the same kind of molecule on the other side o Heterophilic binding: molecules on one cell bind different kind of molecule on the other side o Linker-dependent binding - adhesion molecule son adjacent cells are linked by an extracellular molecule Anchoring junctions: desmosomes (= macula adherens) o Cell-cell-junctions o Molecular composition: • • • • Transmembrane adhesion proteins: desmoglein & desmocolling (cadherin family; disease: pemphigus) • IF: keratin in epithelial cells and desmin in cardiomyocites linked to anchor proteins • Intraellular anchor proteins: plakoglobin & desmoplakin which form dense subplasmalemmal plaques Anchoring Juncions: hemidesmosomes o Cell-matrix o Morphlogically resemble desmosomes o Connect parts of the basal surface of epithelial cells to the underlying basal lamina o Molecular composition: • Transmembrane adhesion proteins: integrins • IF (keratin) linked to anchor protein • Intracellular anchor protein: plectin • Extracellular matrix protein (laminin) linked to integrin Anchorin Junctions: focal adhesion o Cell-matrix o Molecular composition: o Transmembrane adhesion proteins: integrins o AF linked to anchor proteins o Intracellular anchor proteins: α-actinin, talin, vinculin, filamin o Extracellular matrix proteins linked to integrins Integrin structure: Cell-Matrix o Cytoplasmic domain of β-subunit is binding site for the intracellular anchor proteins (talin, filamin, αactinin) that associate with AF. o Ectodomain of α- and β-subunits are binding sites for matrix proteins. Hence integrins are "integrators" of matrix and cytoskeleton (focal adhesions) Anchoring Junctions: Summary of Molecular organization o Cel-cell junctions: • Transmembrane proteins: cadherins • Intracellular anchor proteins: ▪ α-actinin, catenins, vinculin: adherens junctions ▪ Plakoglobin & desmoplakin: desmosomes • Cytoskeletal components linked to anchor proteins ▪ AF: adherens junctions ▪ IF (keratin, desmin): desmosomes o Cell-matrix junctions: • Transmembrane proteins: integrins • Intracellular anchor proteins: ▪ α-actinin, talin, vinculin, filamin: focal adhesions ▪ Plectin: hemidesmosomes • Extracellular matrix proteins (laminin, etc.) Gap junction (nexus, comunicating junction) o Regions of direct intercellular communication o Permit the passage of various small molecules between adjacent cells o The intercellular cleft at the gap junction is narrow and constant, about 2 to 4 nm o The gap is spanned by intercellular channels composed of 2 conneons. Each gap junction contains a cluster of few to many thousands of connexons. Each connexon is formed by 6 connexins - 4 pass transmembrane proteins o Gap juctions can differ in size o Connexons: • Homomeric: assembled by a single type of connexin • Hereomeri: different types of connexins o Justictional channels: • Homotypic • Heterotypic o Gap Junctions allow ions and small molecules including signaling molecules to directly pass from cytoplasm of one cell to the other. Examples: • Electrically excitable cells • Non-electrically excitable cells • Blood glucose • • • • • • Gap junctional channels can flip between open and closed state. Thus, these channels are dynamic structures changing their conformation in response to intracellular and extracellular signal Signal relaying communicating junctions (synapse) Membrane specializations: o Lateral membrane specializations - junctional complexes (TJ, ZO, ZA, desmosomes, gaap j.) o Basal surface specializations - basal lamina, plasma membrane enfoldings, hemidesmosomes Plasma membrane enfoldings: o Increase the surface area available for transport o Partition the mitochondria-rich basal cyoplasm (energy for the transport) o • • • Cell Inclusion • • • • • • • • Materials in the cytoplasm which may or may no be surrounded by a membrane Basic features: o Not present in all cells o Nonmotile and with little or no metabolic activity o Filled with stored macromolecules • Fat droplets • Glycogen granules • Pigments • Crystals Fat droplets o Accumulation of lipid molecules o Prominent in: • Adipocytes (fet cells) • Adrenal cortex vells • Liver cells Glycogen granules o Glycogen is the most common sotrage form of glucose o Especially abundant in cells of muscle and liver o At TEM appears as clusters (rosettes) of β-particles (smaller) or α-particles (larger clusters) that resemble ribosomes, located in the vicinity of the SER o Disorders result in inability to degrade glycogen Glycogen storage disorders: o Result of their inability to degrade glycogen o Major manifestations: • Hepatic • Myopathic • miscellaneous Pigment deposits (PD): o Occur in many cell types and may contain variouus complex substances • Lipofuscin: by-product of lysosomal digestion in long-lived cells • Melanin: protects cell nuclei from damage to DNA by light • Hesmodesiderin granules: containing the protein ferritin, which forms a storage complex for iron Lipofuscin: o Insoluble brownish-yelloww granular intracellular material that accumulates in a variety of tissues (particularly the heart, liver, and brain) as a function of age or atrophy o Complexes of lipid and protein that derive from the free radical-catalyzed peroxidation of polyunsaturated lipids of subcellular membranes o Not inurious to the cell but important as a marker of past free-radical injury Crystals o Found in few cells; probably crystalline forms of certain proteins o Examples: • Sertoli cells of the testis (crystals of Charcot-Böttcher) • Leyding cell of the testis (crytals of Reinke) • Macrophages (sometimes) • Reinke's crystals o Rectangular inclusions (3-20 μm in diameter), composed of protein, in the interstitial cells of the testis (Leydig cells) and hilus cells (analog. to Leydig cells) in the ovary o They are not found in the Leydig cells of non-primate mammals Nucleus • • Cell nucleus: The defining organelle of all eukaryotic cells Parts of the nucleus o Inner + outer nuclear membrane (nuclear envelope) o Perinuclear space o Nuclear pore complex o Nucleoplasm o ER membrane o ER lumen • Functions of the nucleus: o Cellular regulation: Houses genetic material, which directs all cellular activities and regulates cellular structure o Production: Produces ribosomal subunits in nucleolus and exports them into cytoplasm for assembly into ribosomes Nucleus Constituents: o Nuclear envelope (seperates nucleus from cytoplasm) • Inner nuclear membrane (INM) • Outer nuclear membrane (ONM) • Perinuclear space • Nuclear pore complexes (NP) o Nuclear matrix (nucleoplasm) • Chromatin • Nuclear bodies - nuclear compartments • Chromatin • • • Complex of DNA, histones, and nonhistone proteins found in the nucleus of a eukaryotic cell. The material of which chromosomes are made. Euchromatin: o 90% of chromatin o Composed of 30-nm fibers and looped domains Heterochromatin o 10% of chromatin o Involves an additional level of folding of 30-nm fiber and requires many proteins in addition to the histones o Transcriptionally inactive during interphase - gene silencing o Common in centromeres and telomeres of chromosomes