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AS Biology Unit 2 page 1 Heckmondwike Grammar School Biology Department Edexcel A-Level Biology B Contents Cell Theory .............................................................................. p3 Eukaryotic Cells ...................................................................... p4 Prokaryotic Cells .................................................................... p9 Microscopy ............................................................................... p14 Cell Membranes ...................................................................... p19 Transport across Cell Membranes ..................................... p21 Diffusion ........................................................................ p22 Osmosis ........................................................................ p23 Active Transport ......................................................... p26 Bulk Transport ............................................................ p27 These notes may be used freely by biology students and teachers. I would be interested to hear of any comments and corrections. Neil C Millar ([email protected]) June 2016 Y12 Unit 1 Biochemistry Unit 2 Cells Unit 3 Reproduction Unit 4 Transport Unit 5 Biodiversity Unit 6 Ecology Y13 HGS Biology A-level notes Unit 7 Metabolism Unit 8 Microbes Unit 9 Control Systems Unit 10 Genetics NCM/2/16 AS Biology Unit 2 page 2 Biology Unit 2 Specification 2.01 Cells, Tissues and Organs Cell theory is a unifying concept that states that cells are a fundamental unit of structure, function and organisation in all living organisms. In complex organisms, cells are organised into tissues, organs, and organ systems. 2.02 Eukaryotic Cells The ultrastructure of eukaryotic cells and the functions of organelles, including: nucleus, nucleolus, 80S ribosomes, rough and smooth endoplasmic reticulum, mitochondria, centrioles, lysosomes, Golgi apparatus, cell wall, chloroplasts, vacuole and tonoplast. 2.03 Prokaryotic Cells The ultrastructure of prokaryotic cells and the structure of organelles, including: nucleoid, plasmids, 70S ribosomes and cell wall. Distinguish between Gram positive and Gram negative bacterial cell walls. Be able to distinguish between Gram positive and Gram negative bacterial cell walls and why each type reacts differently to some antibiotics. HGS Biology A-level notes 2.04 Microscopy How magnification and resolution can be achieved using light and electron microscopy. The importance of staining specimens in microscopy. 2.05 Cell Membranes and Transport The structure of the cell surface membrane with reference to the fluid mosaic model. How passive transport is brought about by: diffusion; facilitated diffusion (through carrier proteins and protein channels); osmosis. How the properties of molecules affects how they are transported, including solubility, size and charge. Water potential = turgor pressure + osmotic potential. The process of active transport, including the role of ATP. Phosphorylation of ADP requires energy and that hydrolysis of ATP provides an accessible supply of energy for biological processes. Large molecules can be transported into and out of cells through the formation of vesicles, in the processes of endocytosis and exocytosis. NCM/2/16 AS Biology Unit 2 page 3 Blank Page HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 4 Cells Cell Theory All living things are made of cells. Cells were first seen in dead plant tissue in 1665 by Robert Hooke (who named them after monks' cells in a monastery), and living cells were first observed by Leeuwenhoek a few years later using a primitive microscope. However it wasn’t until two centuries later that scientists realised that all living organisms were composed of cells, when Schleiden and Schwann proposed cell theory in 1838. Cell theory states that: 1. All organisms are composed of one or more cells. All the processes of life (e.g. growth, metabolism, reproduction) take place within cells. 2. Cells are the smallest units that can be alive. 3. New cells are always formed by division of old cells and new living cells cannot be spontaneously generated. The first cells must have evolved from non-living structures 4 billion years ago, but the development of “life” took place gradually over millions of years, so there was no definable first cell. Unicellular and Multicellular Organisms Some organisms are made of just a single cell (e.g. bacteria, algae, protozoa, yeast). In these unicellular organisms, the single cell carries out all the process of life. But most organisms are multicellular. They are composed of many cells, which are differentiated to carry out different tasks. Prokaryotic and Eukaryotic Cells Cells can be classified into two large groups: Prokaryotic cells do not have a nucleus, or any interior compartments Eukaryotic cells do have a nucleus, and various other interior compartments, called organelles. We'll examine these two kinds of cell in detail, based on structures seen in electron micrographs. These show the individual organelles inside a cell. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 5 Eukaryotic Cells Eukaryotic cells contain a nucleus and numerous other cell organelles. cell wall small vacuole cell membrane Golgi body cytoskeleton rough endoplasmic reticulum large vacuole chloroplast nucleus mitochondrion nucleoplasm nucleolus nuclear envelope 80S ribosomes smooth endoplasmic reticulum nuclear pore lysosome centriole undulipodium Not all eukaryotic cells have all the parts shown here 10 µm Cell Membrane (or Plasma Membrane) This is a thin, flexible layer round the outside of all cells made of phospholipids and proteins. It separates the contents of the cell from the outside environment, and controls the entry and exit of materials. The structure and function of the membrane is examined in detail on p19. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 6 Nucleus This is the largest organelle. It is roughly spherical and surrounded by a nuclear envelope, which is a double membrane with nuclear pores – large holes containing proteins that control the exit of substances from the nucleus. The interior is called the nucleoplasm, which is full nuclear envelope RER nucleolus of chromatin – the DNA/protein complex. During cell division the observable nuclear pore chromosomes. The nucleolus is a dark region of chromatin, involved nucleoplasm (containing chromatin) chromatin becomes condensed into discrete in making ribosomes. Mitochondrion (pl. Mitochondria) This is a sausage-shaped organelle (8µm long), and is where aerobic outer membrane respiration takes place and ATP is synthesised in all eukaryotic cells inner membrane (anaerobic respiration takes place in the cytoplasm). Cells that use a matrix lot of energy (like muscle cells) have many mitochondria. Mitochondria are surrounded by a double membrane: the outer membrane is simple and quite permeable, while the inner membrane is highly folded into cristae, which give it a large surface area. The space enclosed by the inner membrane is called the mitochondrial matrix, and contains small circular strands of DNA. The inner crista (fold in inner membrane) stalked particles (ATP synthase) ribosomes DNA membrane is studded with stalked particles, which are the enzymes that make ATP. Chloroplast Bigger and fatter than mitochondria, chloroplasts are where photosynthesis takes place, so are only found in photosynthetic organisms (plants and algae). Like mitochondria they are enclosed by a double membrane, but chloroplasts also have a third membrane called the thylakoid membrane. The thylakoid membrane is folded into thylakoid disks, which are then stacked into piles called grana. The space between the inner membrane and the thylakoid is called the stroma. The thylakoid membrane contains chlorophyll and chloroplasts also contain starch grains, ribosomes and circular DNA. outer membrane inner membrane thylakoid membrane granum (thylakoid stack) stalked particles (ATP synthase) starch grain stroma Chloroplasts are the most common type of plastids, which are organelles with double membranes, found in all plant and algae cells. The other types of plastid are not used for photosynthesis, but are usually storage organelles, such as amyloplasts, which store starch. Plastids can multiply inside eukaryotic cells by fission (splitting), just like bacterial cells. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 7 Ribosomes These are the sites of protein synthesis. Ribosomes are the smallest and most numerous of the cell organelles, but, unlike most other organelles, are not enclosed in a membrane. Ribosomes are either found free in the cytoplasm, where they make proteins for the cell's own use, or they are found attached to the rough endoplasmic reticulum, where they make proteins for export from the cell. All eukaryotic ribosomes are of the larger, "80S", type. Endoplasmic Reticulum (ER) This is a series of membrane channels involved in synthesising and transporting materials. Rough Endoplasmic Reticulum (RER) is studded with numerous cisternae ribosomes, which give it its rough appearance. The ribosomes synthesise proteins, which are processed in the RER (e.g. by enzymatically modifying ribosomes the polypeptide chain, or adding carbohydrates), before being exported from the cell via the Golgi Body. Smooth Endoplasmic Reticulum (SER) does not have ribosomes and is used to process materials, mainly lipids, needed by the cell. Golgi Body (or Golgi Apparatus) Another series of flattened membrane vesicles, formed from the endoplasmic reticulum. Its job is to transport proteins destined for extracellular use from the ER to the cell membrane for export. Parts of the RER containing proteins fuse with one side of the Golgi body membranes to form cisternae, while at the other side small vesicles bud off from the cisternae and move towards the cell membrane, where they fuse, releasing their contents to the outside of the cell by exocytosis. Vacuoles These are membrane-bound sacs containing water or dilute solutions of salts and other solutes. Most cells can have small vacuoles that are formed as required, but plant cells usually have one very large permanent vacuole that fills most of the cell, so that the cytoplasm (and everything else) forms a thin layer round the outside. Plant cell vacuoles are surrounded by a membrane called the tonoplast and filled with cell sap. They help to keep the cell rigid, or turgid. Some unicellular protoctists have feeding vacuoles for digesting food, or contractile vacuoles for expelling water. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 8 Lysosomes These are small membrane-bound vesicles formed from the RER containing a cocktail of digestive enzymes. They are used to break down unwanted chemicals, toxins, organelles or even whole cells, so that the materials may be recycled. They can also fuse with a feeding vacuole or a phagosome to digest its contents. Cytoskeleton This is a network of protein fibres extending throughout all eukaryotic cells, used for support, transport and motility. The cytoskeleton is attached to the cell membrane and gives the cell its shape, as well as holding all the organelles in position. The cytoskeleton is also responsible for all cell movements, such as cell division, cilia and flagella, cell crawling and muscle contraction in animals. Centrioles There are always two centrioles found near the nucleus. They are part of the cytoskeleton and are used in cell division to make the spindle fibres that move the chromosomes. Centrioles are found in animal cells, but not in plants, fungi or prokaryotes. Cilia, Flagella and Microvilli These are different finger-like extensions of the cell membrane containing cytoskeleton proteins so they can move. Cilia are short and numerous and are used for moving the cell (e.g. ciliates) or for moving the extracellular fluid (e.g. trachea). Flagella are longer than the cell, there are usually only one or two of them and they are used for motility (e.g. sperm). Microvilli are short extensions found in certain cells such as in the epithelial cells of the intestine and kidney, where they increase the surface area for absorption of materials. Cytoplasm (or Cytosol) This is the solution within the cell membrane. It contains enzymes for glycolysis (part of respiration) and other metabolic reactions together with sugars, salts, amino acids, nucleotides and everything else needed for the cell to function. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 9 Cell Wall This is a thick layer outside the cell membrane. Almost all cells have cell walls, made of different materials: Plant cell walls are made of cellulose Fungal cell walls are made of chitin algal cell walls are made of glycoproteins diatom cell walls are made of silica bacterial cell walls are made of peptidoglycan archaean cell walls are made of S-layer proteins Only animal cells do not have a cell wall. Cell walls are used to give strength and rigidity to cells and to resist osmotic lysis. They are made of a network of fibres that give strength but are freely permeable to solutes (unlike membranes). A wickerwork basket is a good analogy. Plant cell walls are composed of three layers: 1. The middle lamella is a layer of pectins on the outside of the cell wall that glues adjacent cells together. 2. The primary cell wall is a thin layer of cellulose microbfibrils (unit 1), laid down while the cell is growing. The primary cell wall is flexible, so that is can expand as the cell grows. 3. The secondary cell wall is a thick layer built up inside the primary cell wall once the cell has stopped growing. It is made of cellulose microbfibrils (unit 1) embedded in a matrix made of hemicellulose and pectin, and provides most of the strength of the cell. In xylem vessels the secondary cell wall also contains lignin for strength and waterproofing, and these lignified cell walls form woody tissue (unit 4). Plant cell walls are usually studded with channels called plasmodesmata (singular plasmodesma), which link the cytoplasms of adjacent cells, forming a symplast (a continuous cytoplasm, see unit 4). This symplast allows solutes to diffuse freely between cells without crossing any membranes. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 10 Comparison of different types of Eukaryotic Cell Fungi Plants Animals Nucleus Mitochondria Chloroplast 80S ribosome Vacuoles Cytoskeleton Centriole Plasma membrane (chitin) (cellulose) Cell Wall Cell Organisation In multicellular organisms the eukaryotic cells are specialised to perform different functions. Thousands of different types of cell have been described, such as red blood cells, smooth muscle cells, adipose cells, B lymphocytes, osteocytes, motor neurones, ova, ciliated epithelial cells, endocrine cells, hepatocytes, palisade mesophyll cells, guard cells, root hair cells, cambium, and countless more. Eukaryotic cells are arranged into: A tissue is a group of similar cells performing a particular function. Simple tissues are composed of one type of cell, while compound tissues are composed of more than one type of cell. Animal tissues include epithelium (lining tissue), connective, nerve, muscle, blood, glandular. Plant tissues include epidermis, meristem, vascular, mesophyll, cortex. An organ is a group of physically-linked different tissues working together as a functional unit. For example the stomach is an organ composed of epithelium, muscular, glandular and blood tissues. A plant leaf is also an organ, composed of mesophyll, epidermis and vascular tissues. An organ system is a group of organs working together to carry out a specific complex function. Humans have seven main systems: the circulatory, digestive, nervous, respiratory, reproductive, urinary and muscular-skeletal systems. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 11 Prokaryotic Cells Prokaryotic cells are smaller than eukaryotic cells and do not have a nucleus or indeed any membranebound organelles. The prokaryotes comprise the bacteria and archaea (see unit 2). Prokaryotic cells are much older than eukaryotic cells and they are far more abundant (there are ten times as many bacteria cells in a human than there are human cells). The main features of prokaryotic cells are: Cytoplasm. Contains all the enzymes needed for all metabolic reactions, since there are no organelles Ribosomes. The smaller “70S” type, all free in the cytoplasm and never attached to membranes. Used for protein synthesis. Nucleoid. The region of the cytoplasm that contains DNA. It is not surrounded by a nuclear membrane. DNA. Always circular (i.e. a closed loop), and not associated with any proteins to form chromatin. Sometimes referred to as the bacterial chromosome to distinguish it from plasmid DNA. Plasmid. Small circles of DNA, separate from the main DNA loop. Used to exchange DNA between bacterial cells, and also very useful for genetic engineering (see unit 3). Plasma membrane. Made of phospholipids and proteins, like eukaryotic membranes. Cell Wall. This is a thick layer outside the cell membrane used to give a cell strength and rigidity and resist osmotic lysis. Made of peptidoglycan (not cellulose), which is a glycoprotein (i.e. a protein/carbohydrate complex, also called murein). There are two types of cell wall: Gram-positive and Gram-negative (see next page). Capsule. A thick polysaccharide layer outside of the cell wall. Used for sticking cells together, as a food reserve, as protection against desiccation and chemicals, and as protection against phagocytosis. In some species the capsules of many cells fuse together forming a mass of sticky cells called a biofilm. Dental plaque is an example of a biofilm. Flagellum. A rigid rotating helical-shaped tail used for propulsion. The motor is embedded in the cell membrane and is driven by a H+ gradient across the membrane. The bacterial flagellum is quite different from the eukaryotic flagellum. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 12 The Bacterial Cell Wall There are two kinds of bacterial cell wall, which are identified by the Gram Stain technique when observed under the microscope. Gram positive bacteria stain purple, while Gram negative bacteria stain pink. The technique was discovered by Christian Gram in 1884 and is still used today to identify and classify bacteria. We now know that the different staining is due to two types of cell wall: Gram Positive cell wall Gram Negative cell wall (stains purple) (stains pink) Gram positive bacteria have a thick peptidoglycan cell wall outside their cell membrane. The cell wall is very strong and allows these bacteria to withstand severe physical conditions. There may be a capsule outside the cell wall. Gram negative bacteria have a more complex structure, with a thin layer of periplasm (like cytoplasm but outside the cell), a thin peptidoglycan cell wall, and then a second, outer membrane, which contains lipopolysaccharides instead of phospholipids in its outer layer. This layer resists antibiotics and lysozyme enzymes, so gram negative bacteria are more difficult to kill. The Gram Stain A microscope slide is prepared with a sample of the bacteria to be examined. There are four steps in the Gram staining technique: 1. The microscope slide is flooded with crystal violet (the primary stain), which binds to the peptidoglycan cell wall, staining all bacterial cells purple. 2. The slide is flooded with iodine. This binds to crystal violet in the cell wall and fixes it, making it difficult to wash out. 3. The slide is washed with ethanol. The ethanol dissolves the outer lipopolysaccharide membrane of gram-negative cells, whose peptidoglycan cell wall is so thin that the crystal violet is washed out. The peptidoglycan cell wall of gram-positive cells is so thick that the crystal violet cannot be washed out, so these cells remain purple. 4. The slide is flooded with safranin (the counter-stain), which stains the thin peptidoglycan cell wall of gram-negative cells pink. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 13 Bacteria Cells and Antibiotics Antibiotics are antimicrobial chemicals produced naturally by other microbes (usually fungi or bacteria) and used to treat bacterial infections. Antibiotics are so useful because they are selectively toxic i.e. they kill bacteria growing in human tissue without killing the host human cells. Antibiotics do this by inhibiting enzymes that are unique to prokaryotic cells, such those involved in synthesising the bacterial cell wall or 70S ribosomes. For example: penicillin (and related antibiotics ampicillin, amoxicillin and methicillin) Inhibits an enzyme involved in the synthesis of peptidoglycan for bacterial cell wall. This weakens the cell wall, killing bacterial cells by osmotic lysis. The penicillins can’t easily cross cell membranes, so they are only effective against Grampositive bacteria (since the peptidoglycan cell wall is exposed) but not against Gram-negative bacteria (since the wall is behind the outer membrane). Streptomycin, tetracycline and erythromycin inhibit enzymes in 70S ribosomes. This stops protein synthesis so prevents cell division. These antibiotics are equally effective against Gram-positive and Gram-negative bacteria, since they both contain the same 70S ribosomes. These effects are reflected in the concentration of antibiotics required to treat bacterial infections, shown on this chart: HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 14 Summary of the Differences between Prokaryotic and Eukaryotic Cells Prokaryotic Cells Eukaryotic cells small cells (< 5 µm) larger cells (> 10 µm) always unicellular often multicellular no nucleus or any membrane-bound organelles always have nucleus and other membrane-bound organelles DNA is linear and associated with proteins to form chromatin DNA is circular, without proteins ribosomes are small (70S) ribosomes are large (80S) no cytoskeleton always has a cytoskeleton motility by rigid rotating flagellum, made of flagellin motility by flexible waving undulipodium, made of tubulin cell division is by binary fission cell division is by mitosis or meiosis reproduction is always asexual reproduction is asexual or sexual huge variety of metabolic pathways common metabolic pathways Endosymbiosis Prokaryotic cells are far older and more diverse than eukaryotic cells. Prokaryotic cells have probably been around for 3.5 billion years, while eukaryotic cells arose only about 1 billion years ago. It is thought that eukaryotic cell organelles like nuclei, mitochondria and chloroplasts are derived from prokaryotic cells that became incorporated inside larger prokaryotic cells. This idea is called endosymbiosis, and is supported by these observations: organelles contain circular DNA, like bacteria cells. organelles contain 70S ribosomes, like bacteria cells. organelles have double membranes, as though a single-membrane cell had been engulfed and surrounded by a larger cell. organelles reproduce by binary fission, like bacteria. organelles are very like some bacteria that are alive today. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 15 Microscopy Of all the techniques used in biology microscopy is probably the most important. The vast majority of living organisms are too small to be seen in any detail with the human eye, and cells and their organelles can only be seen with the aid of a microscope. Microscopes have two properties: magnification and resolution. Magnification and Resolution Magnification is a dimensionless number, usually written as a magnification factor, e.g. x100. It is simply indicates how much bigger the image is that the original object. By using more lenses, microscopes can magnify by a larger amount, but the image may get more blurred, so this doesn't always mean that more detail can be seen. Resolution is a distance, usually measured in nm. It is the smallest separation at which two separate objects can be distinguished, or resolved. For example if the resolution of a microscope is 50nm, then objects closer together than 50nm cannot be seen as separate objects. The resolution of a microscope is ultimately limited by the wavelength of light used (400-600nm for visible light), so to improve resolution waves with a shorter wavelength is needed. This is how electron microscopes gain resolution. This chart shows the effect of magnification and resolution on the appearance of two small objects. Low resolution (a large distance) High resolution (a small distance) Low magnification High magnification There are three common kinds of microscope: 1. Light (or optical) microscope 2. Transmission electron microscope (TEM) 3. Scanning electron microscope (SEM) HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 16 1. The Light Microscope Light, or optical, microscopes are the oldest, simplest and most widely-used form of microscopy. Specimens are illuminated with visible light, which is focused using glass lenses and viewed using the eye or photographic film. Most light microscopes have three lenses: a condenser lens to focus the light onto the specimen; an objective lens to magnify the image; and an eyepiece lens to focus the image on the eye. The total magnification of a microscope is given by the product of the objective and eyepiece lenses. The resolution of a light microscope is about 200nm, but special optical techniques (like fluorescence microscopy and interference microscopy) can improve this limit down to 1nm. Specimens can be living or dead but need to be thin so that enough light can be transmitted through them. Most biological specimens will then be invisible, so they usually need to be coloured with a chemical stain to make them stand out. Some common stains are: Iodine to stain starch in plant cells Methylene blue or acetic orcein to stain DNA in nuclei Toluidine blue to stain lignin in xylem cell walls Phalloidin to stain the cytoskeleton 2. The Transmission Electron Microscope (TEM) The Transmission Electron Microscope (TEM) uses a beam of electrons, rather than electromagnetic radiation, to "illuminate" the specimen. This may seem strange, but electrons behave like waves and can easily be produced (using a hot wire), focused (using electromagnets) and detected (using a phosphor screen or photographic film). A beam of electrons has an effective wavelength of less than 1nm, so can be used to resolve small sub-cellular ultrastructure. The development of the electron microscope in the 1930s revolutionised biology, allowing organelles such as mitochondria, ER and membranes to be seen in detail for the first time. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 17 Electron microscope specimens need to be very thin, and to prevent them breaking the specimens have to be embedded in plastic. Biological specimens are usually transparent to electrons, so they also have to be stained with electron-dense heavy metal compounds such as gold, osmium or even uranium salts. These stains aren’t coloured, so electron microscope image are always monochrome (though the images can be coloured artificially using computer software). The biggest problem with electron microscopy is that there must be a vacuum inside the electron microscope (or the electron beam would be scattered by the air molecules), so it can't be used for living organisms. Specimens can also be damaged by the electron beam, so delicate structures and molecules can be destroyed. There is always a concern that structures observed by an electron microscope are artefacts (i.e. due to the preparation process and not present in the real cell), but improvements in technique have eliminated most of these. 3. The Scanning Electron Microscope (SEM) The scanning electron microscope (SEM) works in a very different way to other microscopes. It scans a fine beam of electrons onto a specimen and detects the electrons scattered back by the surface. The scattered electron signal is then converted into a computer-generated image of the surface of the specimen. These images are fantastically detailed but do not have very high magnification. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 18 Comparison of Different Microscopes Light Microscope Advantages good magnification (1000x) can use living specimens colour images Limitations poor resolution (200nm) can’t see organelles specimens must be stained TEM high magnification (5 000 000x) very good resolution (1nm) SEM gives 3-dimentional images don’t need thin sections vacuum, so can’t use living specimens resolution not as good as TEM (10nm) complex specimen preparation – specimens need to be very thin and embedded in plastic can’t see internal structures no colour specimens can be damaged by electron beam, causing artefacts. Uses tissues, cells and small organisms cell organelles, prokaryotes surfaces of living and nonand viruses living specimens videos HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 19 Magnification Calculations Microscope drawings and photographs (micrographs) are usually magnified, and you have to be able to calculate the true size of the object from the image. There are two ways of doing this: 1. Using a Magnification Factor Sometimes the image has a magnification factor on it. The formula for the magnification is: For example, if this drawing of an object is 40mm long and the magnification is x1000, then the object's true length is T = I M = 40mm 1000 = 0.04mm = 40μm. Always convert your answer to appropriate units, usually µm for cells and x 1000 organelles. Sometimes you have to calculate the magnification. For example, if this drawing of an object is 40mm long and its true length is 25µm, the magnification of the drawing is: M= I T = 40 000μm 25μm = 1600. Remember, the image and true lengths must be in the same units. Magnifications can also be less than one (e.g. x 0.1), which means that the drawing is smaller than the actual object. 2. Using a Scale Bar Sometimes the picture has a scale bar on it. You can easily judge rough sizes by seeing how many scale bars fit into the object, but to work out a size accurately, you first use the scale bar to calculate the magnification, then you apply this magnification to the object. For example, if this 5µm scale bar is 10mm long, then the magnification is: M= I T = 10 000μm 5μm = 2000. Therefore if this drawing of an object is 40mm long the object's true size is: T = I M = 40mm 2000 = 0.02mm = 20μm. This makes sense, 5µm as we can fit about four scale bars into the object. It's good to have a rough idea of the size of objects, to avoid silly mistakes. A mitochondrion is not 30mm long! Scale bars make this much easier than magnification factors. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 20 The Cell Membrane The cell membrane (or plasma membrane) surrounds all living cells, and is the cell's most important organelle. It controls how substances can move in and out of the cell and is responsible for many other properties of the cell as well. The membranes that surround the nucleus and other organelles are almost identical to the cell membrane. Membranes are composed of phospholipids, proteins and carbohydrates arranged as shown in this diagram. peripheral protein on outer surface carbohydrate attached to protein phospholipid fatty acid chains polar head part of cytoskeleton peripheral protein on inner surface integral protein forming a channel The phospholipids form a thin, flexible sheet, while the proteins "float" in the phospholipid sheet like icebergs, and the carbohydrates extend out from the proteins. This structure is called a fluid mosaic structure because all the components can move around (it’s fluid) and the many different components all fit together, like a mosaic. The phospholipids are arranged in a bilayer (i.e. a double layer), with their polar, hydrophilic phosphate heads facing out towards water, and their non-polar, hydrophobic fatty acid tails facing each other in the middle of the bilayer. This hydrophobic layer acts as a barrier to most molecules, effectively isolating the two sides of the membrane. Different kinds of membranes can contain phospholipids with different fatty acids, affecting the strength and flexibility of the membrane, and animal cell membranes also contain cholesterol linking the fatty acids together and so stabilising and strengthening the membrane. The proteins usually span from one side of the phospholipid bilayer to the other (integral proteins), but can also sit on one of the surfaces (peripheral proteins). They can slide around the membrane very quickly and collide with each other, but can never flip from one side to the other. The proteins have hydrophilic amino acids in contact with the water on the outside of membranes, and hydrophobic amino acids in HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 21 contact with the fatty chains inside the membrane. Proteins comprise about 50% of the mass of membranes, and are responsible for most of the membrane's properties. Transport proteins. Most transport of small molecules across the membrane take place through integral proteins. This transport includes facilitated diffusion and active transport (more details below). Receptor proteins. Receptor proteins must be on the outside surface of cell membranes and have a specific binding site where hormones or other chemicals can bind to form a hormone-receptor complex (like an enzymesubstrate complex). This binding then triggers other events in the cell membrane or inside the cell. Enzymes. Enzyme proteins catalyse reactions in the cytoplasm or outside the cell, such as maltase in the small intestine (more in digestion). Recognition proteins. Some proteins are involved in cell recognition. These are often glycoproteins, such as the A and B antigens on red blood cell membranes. Structural proteins. Structural proteins on the inside surface of cell membranes and are attached to the cytoskeleton. They are involved in maintaining the cell's shape, or in changing the cell's shape for cell motility. Structural proteins on the outside surface can be used in cell adhesion – sticking cells together temporarily or permanently. The carbohydrates are found on the outer surface of all eukaryotic cell membranes, and are attached to the membrane proteins or sometimes to the phospholipids. Proteins with carbohydrates attached are called glycoproteins, while phospholipids with carbohydrates attached are called glycolipids. Remember that a membrane is not just a lipid bilayer, but comprises the lipid, protein and carbohydrate parts. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 22 Movement across Cell Membranes The job of the cell membrane is to control what materials can enter and leave cells. There are five main methods by which substances can move across a cell membrane: Passive transport methods Active transport methods 1. Lipid Diffusion 4. Active Transport 2. Facilitated Diffusion 5. Bulk transport 3. Osmosis (Water Diffusion) Passive Transport Methods Passive processes do not require any energy, other than the thermal energy of the surroundings. Substances move around randomly within cells due to thermal motion, and if there is a concentration difference between two places then the random movement results in a substance diffusing down its concentration gradient from a high to a low concentration. This process is called diffusion. Passive transport is simply diffusion across a cell membrane, so does not require metabolic energy and is simply driven by thermal energy. Substances diffuse in both directions across a membrane, but there is a net movement down a concentration gradient. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 23 1. Lipid Diffusion (Simple Diffusion) A few substances can diffuse directly through the lipid bilayer part of the membrane. The only substances that can do this are hydrophobic (lipid-soluble) molecules such as steroids, and a few extremely small hydrophilic molecules, such as H2O, O2 and CO2. For these molecules the membrane is no barrier at all. Since lipid diffusion is a passive process, no energy is involved and substances can only move down their concentration gradient. Lipid diffusion cannot be switched on or off by the cell. 2. Facilitated Diffusion. Facilitated Diffusion is the diffusion of substances across a membrane through a trans-membrane transport protein molecule. The transport proteins tend to be specific for one molecule, so substances can only cross a membrane that contains an appropriate protein. This is a passive diffusion process, so no energy is involved and substances can only move down their concentration gradient. There are two kinds of transport protein: Channel Proteins form a water-filled pore or channel in the membrane. This allows charged substances to diffuse across membranes. Most channels can be gated (opened or closed), allowing the cell to control the entry and exit of ions. In this way cells can change their permeability to certain ions. Ions like Na+, K+, Ca2+ and Cl- diffuse across membranes through specific ion channels. Carrier Proteins have a binding site for a specific solute and constantly flip between two states so that the site is alternately open to opposite sides of the membrane. The substance will bind on the side where it at a high concentration and be released where it is at a low concentration. Important solutes like glucose and amino acids diffuse across membranes through specific carriers. Sometimes carrier proteins have two binding sites and so carry two molecules at once. This is called cotransport, and a common example is the sodium/glucose cotransporter found in the small intestine (see next page). Both molecules must be present for transport to take place. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 24 3. Osmosis (Water Diffusion) Osmosis is the diffusion of water across a membrane. Water can diffuse across a membrane in two ways: partly through the phospholipid bilayer (an example of lipid diffusion) and partly through protein channels called aquaporins (an example of facilitated diffusion). As we’ve seen, diffusion takes place down a concentration gradient but, as the solvent in all cells, the total concentration of water is very high (about 55mol L-1) and it never changes. However, much of this water is not free to diffuse, since it is bound to solvent molecules as a hydration shell. The more concentrated the solution, the more solute molecules there are in a given volume, and the more water molecules are bound in hydration shells, so the fewer free water molecules there are. Two different solutions can be separated by a cell membrane, since membranes are partially-permeable (i.e. the solvent water molecules can pass through easily, but larger solute molecules cannot). So if there is a concentration difference of solutes across a membrane, there will also be a concentration difference of free water molecules across the membrane, so water will diffuse across. This is osmosis. In osmosis water diffuses from a more dilute solution to a more concentrated solution across a partially-permeable membrane. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 25 Water Potential Osmosis can be quantified using water potential, so we can calculate which way water will move. Water potential is a force and is measured in units of pressure (Pa, or usually kPa), and the rule is that water always "falls" from a high to a low water potential (in other words it's a bit like gravity potential or electrical potential). It is abbreviated by the symbol w or just (the Greek letter psi, pronounced "sy"). Water potential is made up of two components: pressure potential (p) and solute potential (s). water potential The overall pressure on water. can be positive or negative. If two compartments are separated by a partiallypermeable membrane, then water diffuses from the higher to the lower water potential. = = pressure potential p Hydrostatic pressure due to an external force. p is usually positive, which means a compression force (e.g. due to a cell wall) but it can be negative, which means a tension force (e.g. in xylem vessels). Pressure potential used to be known as turgor pressure, and this term is sometimes still applied to plant cells. + + solute potential s Pressure due to dissolved solutes. The higher the solute concentration, the lower the solute potential. s is always negative since pure water has s = 0, and any solute decreases s. Solute potential used to be known as osmotic pressure or osmotic potential, but these terms are obsolete. In this experiment a Visking tube is used a partially permeable membrane that lets water through but not solute molecules. The experiment illustrates the use of the water potential equation. A Visking tube bag containing salt solution is placed in a beaker of pure water. The beaker is open to the air, so there are no external forces on the water, so p=0 and since pure water has s =0 then overall s =0. The Visking bag is floppy (flaccid) so is not generating any pressure so p=0, but the salt solution has a s =-200kPa, so the overall =-200kPa. So water enters the bag by osmosis, down its water potential gradient. As water enters the bag it dilutes the salt, raising s to -100kPa. The incoming water expands the bag till it can expand no more, and it applies a compression force resisting further entry of water (p =+100kPa). At equilibrium the positive pressure potential balances the negative solute potential so the water potential is zero and there is no net movement of water. We can use water potential to understand the behaviour of cells in different surroundings or solutions. There are three situations: A hypotonic solution A solution with a lower solute concentration, or higher s, than the cell. A hypertonic solution A solution with a higher solute concentration, or lower s, than the cell. A isotonic solution A solution with the same solute concentration and s as the cell. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 26 Cells without cell walls (animal cells) Since animal cells don’t have a cell wall, there is rarely an external force acting on them, so p is almost always zero. Animal cells burst in a hypotonic solution (osmotic lysis) and shrink in a hypertonic solution. The crinkled shape is due to the cytoskeleton. Cells with cell walls (plant, fungi and bacteria cells) Cell walls are strong so can apply an external force to the cell, increasing s. In a hypotonic solution plant cells swell until they reach a dynamic equilibrium where the positive pressure potential exactly balances the negative solute potential, so the overall water potential is zero and there is no net movement of water. The cell is turgid and this is the normal state for healthy plant cells. Turgor provides support for leaves and nonwoody stems. In isotonic solutions p=0 and the cells are flaccid. In hypertonic solutions plant cells act like animal cells, shrinking and pulling away from the cell wall. This is called plasmolysis and would kill the cell. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 27 Active Transport Methods and ATP Active processes need energy. All the processes that need energy in a cell (including active transport) use a molecule called adenosine triphosphate (ATP) as their immediate source of energy. ATP is synthesised from ADP and phosphate (Pi) using energy released from glucose in respiration in mitochondria. respiration ADP + Pi active transport ATP The hydrolysis (splitting) of ATP back to ADP and Pi releases energy, which can be used to drive processes like muscle contraction, biosynthesis, and active transport. Active transport therefore involves protein molecules that are ATPase enzymes, which catalyse this hydrolysis of ATP to release the energy. 4. Active Transport Active transport is the pumping of substances across a membrane by a trans-membrane protein pump molecule, using energy from ATP. These transport proteins are ATPase enzymes, and they have an ATPase active site on their cytoplasm side. The protein binds a molecule of the substance to be transported on one side of the membrane, changes shape using energy from ATP splitting, and releases the molecule on the other side. The proteins are highly specific, so there is a different protein pump for each molecule to be transported. Active transport always occurs in the same direction and can transport substances up their concentration gradient. A common active transport pump is the sodium/potassium ATPase (Na/K pump), found in all animal cell membranes. This pump continually uses ATP to actively pump sodium ions out of the cell and potassium ions into the cell. This creates ion gradients across the cell membrane, which can be used to regulate water potential and drive other process. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 28 5. Bulk Transport Membrane proteins can only transport fairly small molecules, like ions, glucose and amino acids. But cells also need to transport large macromolecules like proteins and polysaccharides and even small cells. These large objects are transported by bulk transport using membrane vesicles. The synthesis, movement and fusion of the vesicles require metabolic energy in the form of ATP, so this is another active transport process. Transport into a cell is called endocytosis and transport out of a cell is called exocytosis. Endocytosis A part of the membrane folds to form a pocket that deepens and pinches shut to form a vesicle containing whatever material was captured outside the cell. Endocytosis of large particles in suspension (such as microbial cells, viruses, dust particles and cellular debris) is called phagocytosis (“cell eating”). Phagocytosis is used by large white blood cells called phagocytes to engulf and destroy pathogens in the blood (unit 8). They also recycle dead and damaged red blood cells. The vesicle, called a phagosome, often fuses with a lysosome so that the lysozyme enzymes can digest the engulfed particles into small soluble molecules. These molecules are then recycled for cell growth. Endocytosis of small volumes of extra-cellular fluid with its dissolved solutes is called pinocytosis (“cell drinking”). Many unicellular protoctists feed this way, and human cells take up cholesterol-containing lipoproteins (LDLs) from the blood by pinocytosis. Exocytosis This is the reverse of endocytosis. A membrane vesicle fuses with the cell membrane so that its contents are released to the outside. The vesicle is usually formed from the endoplasmic reticulum and the Golgi apparatus, where proteins, lipids and other substances are synthesised and processed for export. Exocytosis is used by secretory cells to secrete hormones into the blood and digestive enzymes into the intestine, and by nerve cells to release neurotransmitter chemicals at synapses (Y13). Exocytosis is also used by plant cells to export the proteins and carbohydrates needed to make the cell wall. HGS Biology A-level notes NCM/2/16 AS Biology Unit 2 page 29 Effect of concentration difference on rate of transport The three kinds of transport can be distinguished experimentally by the effect of solute concentration on its rate of transport: Lipid diffusion shows a linear relationship. The greater then concentration difference the great the rate of diffusion (see Fick’s law, unit 4)). Facilitated diffusion has a curved relationship with a maximum rate. At high concentrations the rate is limited by the number of transport proteins. Active transport has a high rate even when there is no concentration difference across the membrane. Active transport stops if cellular respiration stops, since there is no ATP. Summary of Membrane Transport method uses energy? which part of membrane? specific? concentration gradient Lipid Diffusion phospholipid bilayer Osmosis phospholipid bilayer Facilitated Diffusion proteins Active Transport proteins HGS Biology A-level notes NCM/2/16