Biomembranes and internal cell Organization RNDr. Jan Škoda, Ph.D. Department of Experimental Biology Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Memory system / All types of cells -System of nucleic acids and proteins (storage and expression of genetic information) Membrane system / All types of cells - quantitative differences -System of biomembranes (flow of matter, energy and information) Cytoskeletal system / Eukaryotes, analogy in prokaryotes -System of filamentous protein structures (motility, spatial organization, communication) 2 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) 111 Outline -Structure and properties of biomembranes - Plasma membrane - Biomembranes in prokaryotic cells -Compartmentalization of eukaryotic cells - Membrane fusion and vesicular transport 3 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Structure and properties of biomembranes - Biomembranes and internal cell organization (16 Mar 2022) The biomembrane - Lipid bilayer with embedded proteins Thylakoid membrane - Fluid character - allows movement of molecules -5-7.5 nm thick - Prokaryotes Outer membrane - Plasma membrane, Cyanobacteria - thylakoids, membrane vesicles (further reading) - ! Bacteria: mesosomes - invaginations of the plasma membrane (fixation artifacts?) - ! Cyanobacteria: aerotopes (gas vacuoles; formed by a single layer of Iprotein!) - Eukaryotes - Plasma membrane, membrane organelles, membrane transport vesicles - Compartmentalization 5 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Lipid bilayer - Universal and essential structure of biomembranes Hydrophilic heads Hydrophobic tails polar group phosphate glycerol IS IS IB IB e s phospholipid molecule 6 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) 51445541 7 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Lipid bilayer -General structure of a membrane lipid: 1 hydrophilic head, 1-2 hydrophobic tails 3 main types of membrane lipids: - Phospholipids - most abundant -Sterols - regulation of fluidity -Glycolipids - stability, recognition -Asymmetric composition: differences in the abundance of individual lipids in inner and outer layer of the bilayer 8 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) 111 Phospholipid Glycolipid Cholesterol Phospholipids -(a) glycerolphospholipids (b) sphingophospholipids Major mammalian phospholipids (most plasma membrane mol.): a) glycerol-containing polar (hydrophilk) head group nonpolar (hydrophobic) tails cis-double bond b) derived from sphingosine OH OH I I HC — CH—CH, I'm CH NH30 sphingosine CH3 CH3 ©NH3 ©NH3 CH3^I^CH3 ©f CH3^I^-CH3 © T L CH2 H—C—COO © 1 F r CH2 CH2 CH2 0 1 0 1 0 0 0 = P—0 © 1 0 = P—0 © 1 0 = P—0 © o=p—0 © 1 0 0 1 0 OH 0 CH2—CH —CH2 O O I I c=o c=o I I phosphatidyl-ethanolamine I CH2— CH —CH2 O O I I C=0 c=o So© u u * t n phosphatidyl- I CH2—CH—CH2 O O I I c=o c=o Ě < Ě a 5 5 Li. li_ |\ phosphatidylcholine I I CH —CH—CH2 I I CH NH II I CH C=0 i I 8 A sphingomyelin 9 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Sterols - Do not form the bilayer themselves -Cholesterol - bidirectional regulation of membrane fluidity Glycolipids - Lipids with attached carbohydrate groups (sugar) -Appear on the membrane layer that faces extracellular space Outside the Cell Carbohydrate Plasma Membrane Glycoprotein Glycolipid Protein Inside the Cell OH H Ceramide Galactose galactocerebroside 11 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Lipids are amphiphilic - Polar (hydrophilic) and non-polar (hydrophobic) -> packing in an aqueous environment -Self-sealing nature of biomembranes Spontaneously form closed structures: 12 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Liposomes synthetic vesicles shape of lipid packing of lipid liposome - Micelles remnants of a single lipid leaflet Fluidity of a lipid bilayer - Continuous movement of the lipids Frequent within a monolayer (leaflet) - Rotation - Lateral diffusion Rare between the monolayers - Flip-flop - enzymes can catalyze rapid flip-flop: flippase, floppase, scramblase lateral diffusion Flippase flexion rotation Floppase 13 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Membrane proteins - Integral monotypie proteins - Localized at one side of the bilayer (span only one leaflet) - Most often bound by covalent bonds -Transmembrane proteins (integral polytypic) - Span the bilayer (even multiple times) Hydrophobic polypeptide regions inserted within the bilayer Hydrophilic regions may be present at both sides of the bilayer - Peripheral membrane proteins - Noncovalent interactions with integral proteins Integral Proteins Peripheral Proteins (transmembrane) (monotopic) Peripheral Proteins 14 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Essential biomembrane functions -Creates a barrier between inner and outer environment - Regulates communication between the cell and surrounding environment: transport of molecules and information (signaling) -Cell motility and growth (animal cells) - Intracellular compartmentalization (eukaryotes) Organelles with different environment (pH, concentration of molecules) can cooperate in one cell 16 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) fl Membrane transport (protein-free bilayer) Semipermeable - different permeability to classes of molecules: Small nonpolar molecules - free diffusion Small uncharged polar molecules - slow diffusion; H20 - water channels/aquaporins Large uncharged polar molecules - impermeable Ions - highly impermeable HYDROPHOBIC MOLECULES SMALL UNCHARGED POLAR MOLECULES LARGE UNCHARGED POLAR MOLECULES synthetic lipid bilayer 17 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Gab« Hydrophobie Small polar Large polar Charged molecules Bi1700en Cell Biology/ 03 - Biomembranes and internal cell organization (16 Mar 2022) 394361^9754934^90119617109 Membrane transport mechanisms - Diffusion: passive transport, a substance follows concentration gradient -Channel-mediated: passive transport, closed/open conformations -Transporter-mediated: passive or active, conformational change mediates transport Endocytosis/exocytosis - transport using membrane vesicles lipid bilayer transported molecule channel protein protein transporter i i intr idic \ concentration gradient simple channel-diffusion mediated transporter-mediated PASSIVE TRANSPORT ACTIVE TRANSPORT 19 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Channel-mediated transport CLOSED OPEN lipid bilayer II Ion channels - Ion selectivity -Gating - closed/open conformation regulated by different mechanisms selectivity filter in aqueous pore OPEN Aquaporins / bacterial water channels - Rapid passage of water molecules 20 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) voltage-gated CLOSED I II ligand-gated (extracellular ligand) \ * r ii 4 Laj... w u ligand-gated (intracellular ligand) 1 I 4 w % mechanically gated ft ^^CYTOSOL Transporter-mediated transport solute Passive (downhil - Binding of a solute induces conformation changes Active (uphill) - Primary: ATP-driven or light-driven pumps -Secondary: symport vs. antiport ■ Coupled transport; e.g. symport of glucose by Na+ gradient lipid bilayer oof •© Electrochemical gradient concentration gradient 4 transporter mediating solute-binding site passive transport ^Ca++ H-^ Li9ht Na++ Glucose HCO3 S H <>WS?S interactions (e.g., E-cadherin) (E) tight junctions (specialized intercellular junctions; e.g., in entorocytes) apical plasma membrane lateral plasma membrane basal plasma membrane 29 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Functions of membrane proteins Microfilaments of cytoskeleton CYTOPLASM Attachmentto the cytoskeleton and extracellular matrix (ECM) Signal transduction 30 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Osmosis OSMOSIS The movement of solvent molecules cats through a membrane into a region of hiqhor solute lower cat concentration. 31 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Osmosis -Semipermeable plasma membrane -Solvent (water) can freely enter the cell (diffusion & !aquaporins/water channels) -Solutes mostly impermeable -Solvent tends to equalize the solute concentrations on the two sides of the plasma membrane - Osmotic effects 32 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Osmotic effects Osmotic gradient (concentration of solute inside vs. outside the cell) Hypotonic solution (>) - Water enters the cell, increased volume - Animal cells: plasmoptysis/osmotic lysis (in erythrocytes osmotic hemolysis) Hypertonic solution (<) ■ Cell loses water, reduced volume of cytoplasm - Animal cells: plasmorhisis (shrinkage) - Plant cells: plasmolysis Isotonic solution (=) ■ Water passes the membrane in both directions Hypotonic solution H,0 Isotonic solution Hypertonic solution Animal cell Lysed Ho0 Plant cell Turgid (normal) 33 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Red blood cells under different osmotic pressure echinocytes spherocytes 34 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Biomembranes in prokaryotes ology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Prokaryotic cells - Plasma membrane covered by cell wall (peptidoglycan) Gram-negative bacteria -Additional outer membrane, periplasmic space Gram-positive bacteria, Archea -Single plasma membrane - Bacteria: thicker cell wall -Archea: different cell wall composition - pseudopeptidoglykan 36 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Gram-positive bacterial cell wall Lipoteichoic acid Cell wall (LTA) Gram-negative bacterial cell wal Phospholipid (fatty acid D-glycerol esters) Outer membrane protein I I } Peptidoglycan Cytoplasmic membrane Lipopolysaccharide (LPS) Outer membrane Lipoprotein p , . r-r^-J H H H H I- Periplasms space Cytoplasmic membrane protein Cytoplasmic membrane protein Archaeal cell wall (Methanobacterium, Methanosphaera, Methanobrevibacter) Pseudopeptidoglycan (pseudomurein) Phospholipid -* (isopranoid alcohol L-glycerol ethers) Heteropolysaccharide Archaeal cell wall {Methonococcus, Halobacterium) Phospholipid _«. (isopranoid alcohol L-glycerol ethers) I Cytoplasmic membrane protein S-layer Cytoplasmic membrane Cytoplasmic membrane protein Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Cyanobacteria -Thylakoids "photosynthetic membranes" 38 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Cyanobacteria -Thylakoids - membrane compartments with photosynthetic pigments: phycobilins, carotenoids, various forms of chlorophyll plasma memtxane (4-5 nm) Periplasms Peptidoglycan Space layer Plasma 0uter \ Membrane Membrane phycobilisome thylakoid membrane Thylakoid Membranes Photosystem 1 ™ Photosystem I Photosystem II 39 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Compartmentalization of eukaryotic cells 03 - Biomembranes and internal cell organization (16 Mar 2022) chloroplast ribosomes rough endoplasmic reticulum smooth endoplasmic reticulum nucleoplasm nucleolus nuclear envelope nuclear pore =3 CD r-0 cell wall plasmodesma vesicle cell membrane Golgi apparatus 41 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) 42 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) nuclear envelope nucleus 43 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Animal cell Plant cell Fungal cell Plasma membrane Plasma membrane Plasma membrane Glycocalyx Cell wall Cell wall Nucleus Nucleus Nucleus Endoplasmic reticulum Endoplasmic reticulum Endoplasmic reticulum Golgi apparatus Golgi apparatus Golgi apparatus Lysosomes Vacuoles Vacuoles Peroxisomes Peroxisomes Peroxisomes - Glyoxysomes Glyoxysomes Mitochondria Mitochondria Mitochondria - Chloroplasts - Borders of the cell, interaction with its surrounding environment Catabolism Storage and expression of the genetic information, anabolism Energy metabolism, apoptosis 44 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Brief overview of cellular compartments and their function - Cytosol: protein synthesis, signaling and metabolic pathways - Nucleus: genome, DNA and RNA synthesis ■ Endoplasmic reticulum (ER): lipid synthesis, synthesis of secreted and integral membrane proteins, Ca2+ regulation ■ Golgi apparatus: posttranslational modification of proteins, modifications of lipids, cargo sorting to the secretory pathway - Mitochondrion: ATP synthesis (OXPHOS) - Chloroplasts: ATP synthesis and carbon fixation (photosynthesis) ■ Lysosomes/vacuoles: degradation of molecules / organelles (autophagy), turgor in plants/fungi ■ Peroxisomes, glyoxysomes: oxidation of long chain fatty acids (p-oxidation), detoxification of various harmful compounds - Membrane vesicles: transport of cargo between organelles/to PM, endosomes, exosomes... 45 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) fl Nucleus -Central organelle, spherical shape, (missing in mammalian RBC) -Genome - chromatin (DNA+histones), replication, transcription - Nuclear envelope - Double-membrane structure: outer membrane - perinucler space - inner membrane ■ Contains nuclear pores (transport of larger molecules) ■ Continues into ER - perinuclear space joined with the lumen of ER Inner membrane interacts with nuclear lamina and chromatin Nucleolus - Most commonly single, but several possible in one nucleus - rRNA genes, nascent ribosome assembly Nuclear envelope Chromatin (condensed) Nucleolus Nuclear pores Nucleus Cisternae 46 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Nuclear envelope - ER, nuclear pores and lamina Nucleolus - rRNA transcription & ribosome assembly Translation of ribosomal protein mRNAs Recycling of shuttling factors Mature 40S 2 [1.ITI Nucleolus Nucleoplasm envelope | | Cytoplasm ^0 U3snoRNP # elF6 ^ ITS2 O* 5S rRNA 9 Nuclear transport factors V Ran GTPase 9 Arxl-Albl £ Rixl complex ^^^Real (£ Syo1 Nog2 Non-ribosomal biogenesis factors O 40S ribosomal proteins 9 60S ribosomal proteins Nuclear pore complex 48 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Endoplasmic reticulum (ER) - Network of cisternae and tube-like membrane structures, joined with perinuclear space -Rough ER: cisternae with attached ribosomes, protein synthesis and posttranslational modifications -Smooth ER: tubes w/o ribosomes, synthesis of lipids, phospholipids, steroids Calcium (Ca2+) storage and homeostasis ■ Ca2+ signaling; mitochondria overload - apoptosis 49 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) fl 50 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) ER nucleus nuclear envelope p endoplasmic reticulum nuclear envelope rough ER vesicular tubular clusters c/s Golgi network 1 jxm 51 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Golgi apparatus (GA) - Network of membrane cisternae and membrane-bound vesicles - Modifications of proteins and lipids, packaging into vesicles: sorting for their transport Functional polarization: - c/s face (facing ER) vs trans face -several Golgi stacks per cell ■ Animal cells: up to 100; plant cells: different distribution Golg apparatus Secretory -(fj) vesicle Plasma membrane • Transport vesicle Golgi stack ■ trans face 52 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Golgi apparatus (GA) v 6 Mitochondrion - Energy metabolism - oxidative phosphorylation (ATP synthesis) 2 biomembranes - 2 compartments -Outer membrane i-2 urn-1 Outer membrane DNA Inner membrane - Inner membrane: cristae Respiratory chain proteins - ATP synthase - Intermembrane space ■ Cytochrome c: ETC & apoptosis - Matrix - Enzymes of metabolic pathways Cristae Matrix space Intermembrane space ATP synthase Transporter. ^ Si Creatine * kinase O c3 Enzymes of oxidative metabolism Respiratory chain Matrix space ^o^S o r ®P Nucleot J o ° fr VJ O inter-Inner /"sO membrane >* membraneCv space fj- Enzymes of lipid metabolism Porin Outer membrane 54 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Mitochondria: semiautonomous and dynamic -Own genome (circular mtDNA) and protein synthesis apparatus - Mitochondrial ribosomes similar to prokaryotic —► endosymbiosis -Highly dynamic - Fusion / fission - Mitochondrial network/fragmented - Associated with microtubules - Biogenesis only from pre-existing mitochondria - Autoreplication capacity Mitochondria Endoplasmic reticulum and mitochondria interact to form microenvironments for the assembly of DRP1, MFF, and Bax. Mitophagy (degradation) Fission: increase in mitochondrial numbers Endoplasmic reticulum 55 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Mitochondria form mitochondrial network - Mitochondrion - Mitochondrial network 100 ran 56 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Cristae: oxidative phosphorylation 57 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Chloroplasts - Energy metabolism: photosynthesis (ATP synthesis and carbon fixation); only in plant cells 2 biomembranes - 2 compartments -Outer membrane - intermembrane space - inner membrane -stroma with thylakoids containing photosynthetic pigments and enzymes -Own genome (circular cpDNA) and protein synthesis apparatus -Chloroplast ribosomes similar to prokaryotic —► endosymbiosis - Autoreplication capacity 58 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Chloroplasts CHLOROPLAST LEAF upper epidermis lower epidermis outer membrane thylakoid' membrnae Inner membrane GRANUM thylakold space intermembrane space 59 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Lysosomes -Catabolism: degradation of molecules and organelles -Acidic interior: -50 different pH-sensitive hydrolases, pH -4.5-5.0 - Highly glycosylated membrane proteins - protection against degradation by hydrolases - Primary (newly formed) vs. secondary (fusion of the primary lysosome with an endosome/phagosome) - Extracellular digestion - release of lysosomal enzymes outside the cell (e.g., bone resorption; crucial balance) 60 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) fl Vacuoles - Functional analogy of lysosomes, in plant and fungal cells -Catabolism: degradation of molecules - Storage of water - regulation of turgor 61 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Peroxisomes, glyoxysomes -Catabolism: long fatty acid oxidation, oxidation of toxic molecules - Detoxification of reactive oxygen species (H202, superoxide) - Derived from ER, membrane growth and division - Contain oxidases, catalase Peroxisome (2H202^2H20+02), luciferase (in fireflies) ■ Glyoxysomes: specialized peroxisomes in plant cells (e.g., seeds that contain fats and oils) and fungal cells 62 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) fl If organelles could fa Ik t£eie Hie BiobcxiS+- 63 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Membrane fusion and vesicular transport 64 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) A fundamental process in life Examples of membrane fusion Animal cell Plasma-membrane repair Viral entry Yeast cell Regulated exocytosis ^Golgi ER. <0 Endosome-lysosome /^~\ fusion / J Endosome fusion Cell-cell fusion site Cell-cJA fusion s«e u Vacuole fusion • SNAREs O Tethering/regulatory factors O Dysferlin, myoferlin. tricalbin O Ig-domain proteins A Viral fusion protein O Mitofusin/Fzol • OPAl/Mgml ■ HOPS O Synaptotagmin "*s" Actin bundles 65 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Cell-cell fusion Natural fusion - Fertilization - Formation of syncytium - Yeast mating: fusion of haploid cells Induced fusion - use of fusogens: - Electric field (electroporation) - Chemical (e.g., polyethylenglycol, aka PEG) - Viruses (e.g., Sendai virus) / \ Virus / \ S ~A (o]*(oJ—(q?j 66 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Cell-cell fusion as an evidence of membrane fluidity mouse cell rhodamine labeled membrane protein fluorescein labeled membrane protein hybrid cell NCUBATION AT 37°C time = 0 minutes after cell fusion time = 40 minutes after cell fusion 1970, Frye-Eddidin experiment Lateral diffusion of proteins human cell 68 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Essential roles of membrane fusion - Formation of membrane-bound vesicles and their trafficking between organelles: cargo transport, secretion, uptake Organelle dynamics (e.g., mitochondria) late endosome < Endocytosis Exocytosis 70 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022) Membrane fusion in action Watch video of amoeba phaqocvtosinq other protists 71 Bi1700en Cell Biology / 03 - Biomembranes and internal cell organization (16 Mar 2022)