Transmission electron microscopes (TEMs) are used mainly to study the internal ultrastructure of cells.
A TEM aims an electron beam through a thin section of the specimen.
The image is focused and magnified by electromagnets.
To enhance contrast, the thin sections are stained with atoms of heavy metals.
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1A Tour of the CellsMicroscopyThe discovery and early study of cells improved with the invention of microscopes in the 17th century.In a light microscope (LM) visible light passes through the specimen and then through glass lenses.The lenses refract light such that the image is magnified into the eyeMicroscopesMagnification is the ratio of an object’s image to its real size.Resolving power is a measure of image clarity.It is the minimum distance two points can be separated by and still be viewed as two separate points.MicroscopesLight microscopes can magnify effectively to about 1,000 times the size of the actual specimen.At higher magnifications, the image blursMicroscopesWhile a light microscope can resolve individual cells, it cannot resolve organelles.To resolve smaller structures we use an electron microscope (EM), which focuses a beam of electrons through the specimen or onto its surface.MicroscopesTransmission electron microscopes (TEMs) are used mainly to study the internal ultrastructure of cells.A TEM aims an electron beam through a thin section of the specimen.The image is focused and magnified by electromagnets.To enhance contrast, the thin sections are stained with atoms of heavy metals.TEMScanning electron microscopes (SEMs) are useful for studying surface structures.The sample surface is covered with a thin film of gold.The beam excites electrons on the surface.These secondary electrons are collected and focused on a screen.The SEM has great depth of field, resulting in an image that seems three-dimensional.SEMElectron microscopes reveal organelles, but they can only be used on dead cells Light microscopes do not have as high a resolution, but they can be used to study live cells.Microscopes are a major tool in cytology, the study of cell structures.Cytology coupled with biochemistry, the study of molecules and chemical processes in metabolism, developed into modern cell biology.Electron MicroscopesThe goal of cell fractionation is to separate the major organelles of the cells so that their individual functions can be studied.Isolating Cell OrganellesThis process is driven by an ultracentrifuge, a machine that can spin at up to 130,000 revolutions per minuteFractionation begins with homogenization, gently disrupting the cell.Then, the homogenate is spun in a centrifuge to separate heavier pieces into the pellet while lighter particles remain in the supernatant.Repeating the process for longer & faster collects smaller organelles in the pelletCell FractionationFacts About CellsCells are the basic living units of organization and functionAll cells come from other cellsWork of Schleiden, Schwann, and Virchow contributed to this theoryEach cell is a microcosm of lifeCell TheoryBiological size and cell diversityCell surface area-to-volume ratioPlasma membrane must be large enough relative to cell volume to regulate passage of materialsVolume increases faster than surface area so cell must DIVIDECell size and shape related to functionCell SizeAll cells are surrounded by a plasma membrane.The semifluid substance within the membrane is the cytosol, containing the organelles.All cells contain chromosomes which have genes in the form of DNA.All cells also have ribosomes, tiny organelles that make proteins using the instructions contained in genes.Prokaryotes & EukaryotesA major difference between prokaryotic and eukaryotic cells is the location of chromosomes.In a eukaryotic cell, chromosomes are contained in a membrane-enclosed organelle, the nucleus.In a prokaryotic cell, the DNA is concentrated in the nucleoid region without a membrane separating it from the rest of the cell.Prokaryotes & EukaryotesPROKARYOTEIn eukaryote cells, the chromosomes are contained within a membranous nuclear envelope.The region between the nucleus and the plasma membrane is the cytoplasm.All the material within the plasma membrane of a prokaryotic cell is cytoplasm.Prokaryotes & EukaryotesWithin the cytoplasm of a eukaryotic cell is a variety of membrane-bounded organelles of specialized form and function.These membrane-bounded organelles are absent in prokaryotes.Prokaryotes & EukaryotesEukaryotic cells are bigger than prokaryotic cellsAbility to carry on metabolism set limits on cell sizeApproximate Cell Size:Smallest bacteria, mycoplasmas between 0.1 to 1.0 micronMost bacteria are 1-10 microns in diameter, while Eukaryotic cells are typically 10-100 microns in diameterProkaryotes & EukaryotesThe plasma membrane functions as a selective barrier that allows passage of oxygen, nutrients, and wastes for the whole volume of the cell.The volume of cytoplasm determines the need for this exchange.Rates of chemical exchange may be inadequate to maintain a cell with a very large cytoplasm.The need for a surface sufficiently large to accommodate the volume explains the microscopic size of most cells.Larger organisms do not generally have larger cells than smaller organisms - simply more cells.Importance of Surface AreaA eukaryotic cell has extensive and elaborate internal membranesPartition the cell into compartmentsMany enzymes are built into membranesMembrane compartments are involved in many METABOLIC functionsInternal MembranesThe general structure of a biological membrane is a double layer of phospholipids with other lipids and diverse proteins.Each type of membrane has a unique combination of lipids and proteins for its specific functions.For example, those in the membranes of mitochondria function in cellular respiration.Membrane StructureNucleus & RibosomesContains most of the genes in a eukaryotic cell.Some genes are located in mitochondria and chloroplasts.Averages about 5 microns in diameter.Separated from the cytoplasm by a double membrane.These are separated by 20-40 nm.Where the double membranes are fused, a pore allows large macromolecules and particles to pass through.NucleusThe nuclear side of the envelope is lined by the nuclear lamina, a network of intermediate filaments that maintain the shape of the nucleus. Within the nucleus, the DNA and associated proteins are organized into fibrous material, chromatin.In a normal cell they appear as a diffuse mass. However when the cell prepares to divide, the chromatin fibers coil up to be seen as separate structures, chromosomes.Each eukaryotic species has a characteristic number of chromosomes.A typical human cell has 46 chromosomes, but sex cells (eggs and sperm) have only 23 chromosomes. In the nucleus is a region called the nucleolus.In the nucleolus, ribosomal RNA (rRNA) is synthesized and assembled with proteins from the cytoplasm to form ribosomal subunits.The subunits pass from the nuclear pores to the cytoplasm where they combine to form ribosomes.In the nucleus is a region called the nucleolus.In the nucleolus, ribosomal RNA (rRNA) is synthesized and assembled with proteins from the cytoplasm to form ribosomal subunits.The subunits pass from the nuclear pores to the cytoplasm where they combine to form ribosomes.The nucleus directs protein synthesis by synthesizing messenger RNA (mRNA).The mRNA travels to the cytoplasm and combines with ribosomes to translate its genetic message into the primary structure of a specific polypeptide.Ribosomes contain rRNA and protein.A ribosome is composed of two subunits that combine to carry out protein synthesis.RibosomesCell types that synthesize large quantities of proteins (e.g., pancreas) have large numbers of ribosomes and prominent nuclei.Some ribosomes, free ribosomes, are suspended in the cytosol and synthesize proteins that function within the cytosol.Other ribosomes, bound ribosomes, are attached to the outside of the endoplasmic reticulum.These synthesize proteins that are either included into membranes or for export from the cell.Ribosomes can shift between roles depending on the polypeptides they are synthesizing.Endomembrane SystemMany of the internal membranes in a eukaryotic cell are part of the endomembrane system.These membranes are either in direct contact or connected via transfer of vesicles, sacs of membrane.In spite of these links, these membranes have diverse functions and structures. The endomembrane system includes the nuclear envelope, endoplasmic reticulum, Golgi apparatus, lysosomes, vacuoles, and the plasma membrane.The endoplasmic reticulum (ER) accounts for half the membranes in a eukaryotic cell.The ER includes membranous tubules and internal, fluid-filled spaces, the cisternae.The ER membrane is continuous with the nuclear envelope and the cisternal space of the ER is continuous with the space between the two membranes of the nuclear envelope.Endoplasmic ReticulumThere are two connected regions of ER that differ in structure and function.Smooth ER looks smooth because it lacks ribosomes.Rough ER looks rough because ribosomes (bound ribosomes) are attached to the outside, including the outside of the nuclear envelope. The smooth ER is rich in enzymes and plays a role in a variety of metabolic processes.Enzymes of smooth ER synthesize lipids, including oils, phospholipids, and steroids.These includes the sex hormones of vertebrates and adrenal steroids.The smooth ER also catalyzes a key step in the mobilization of glucose from stored glycogen in the liver.Other enzymes in the smooth ER of the liver help detoxify drugs and poisons.Also detoxifies alcohol and barbiturates.Frequent exposure leads to the proliferation of smooth ER, increasing tolerance to the target and other drugs.Rough ER is especially abundant in those cells that secrete proteins.As a polypeptide is synthesized by the ribosome, it is threaded into the cisternal space through a pore in the ER membrane.Many of these polypeptides are glycoproteins, polypeptides to which an oligosaccharide is attached.Rough ER is especially abundant in those cells that secrete proteins.As a polypeptide is synthesized by the ribosome, it is threaded into the cisternal space through a pore formed by a protein in the ER membrane.Many of these polypeptides are glycoproteins, polypeptides to which an oligosaccharide is attached.These secretory proteins are packaged in transport vesicles that carry them to their next stage.Rough ER is also a membrane factory.Membrane bound proteins are synthesized directly into the membrane.Enzymes in the rough ER also synthesize phospholipidsAs the ER membrane expands, parts can be transferred as transport vesicles to other components of the endomembrane system. Many transport vesicles from the ER travel to the Golgi apparatus for modification of their contents.The Golgi is a center of manufacturing, warehousing, sorting, and shipping.The Golgi apparatus is especially extensive in cells specialized for secretion. Golgi ApparatusThe Golgi apparatus consists of flattened membranous sacs – cisternae (looks like a stack of pita bread)The membrane of each cisterna separates its internal space from the cytosolOne side of the Golgi, the cis side, receives material by fusing with vesicles, while the other side, the trans side, buds off vesicles that travel to other sites. During their transit from the cis to the trans pole, products from the ER are modified to reach their final state.This includes modifications of the oligosaccharide portion of glycoproteins.The Golgi can also manufacture its own macromolecules, including pectin and other noncellulose polysaccharides.During processing material is moved from cisterna to cisterna, each with its own set of enzymes.Finally, the Golgi tags, sorts, and packages materials into transport vesicles.The lysosome is a membrane-bounded sac of hydrolytic enzymes that digests macromolecules.LysosomesLysosomal enzymes can hydrolyze proteins, fats, polysaccharides, and nucleic acids.These enzymes work best at pH 5.Proteins in the lysosomal membrane pump hydrogen ions from the cytosol to the lumen of the lysosomes.While rupturing one or a few lysosomes has little impact on a cell, massive leakage from lysosomes can destroy an cell by autodigestionUsed to destroy old cells; called “CELL DEATH”The lysosomal enzymes and membrane are synthesized by rough ER and then transferred to the Golgi.At least some lysosomes bud from the trans face of the Golgi. Lysosomes can fuse with food vacuoles, formed when a food item is brought into the cell by phagocytosis.As the polymers are digested, their monomers pass out to the cytosol to become nutrients of the cell.Lysosomes can also fuse with another organelle or part of the cytosol.This recycling,or autophagy,renews the cell. The lysosomes play a critical role in the programmed destruction of cells in multicellular organisms.This process allows reconstruction during the developmental process.Several inherited diseases affect lysosomal metabolism.These individuals lack a functioning version of a normal hydrolytic enzyme.Lysosomes are engorged with indigestable substrates.These diseases include Pompe’s disease in the liver and Tay-Sachs disease in the brain. Vesicles and vacuoles (larger versions) are membrane-bound sacs with varied functions.Food vacuoles, from phagocytosis, fuse with lysosomes.Contractile vacuoles, found in freshwater protists, pump excess water out of the cell.Central vacuoles are found in many mature plant cells.Vacuoles have Diverse Functions in Cell MaintenanceCentral VacuoleThe membrane surrounding the central vacuole, the tonoplast, is selective in its transport of solutes into the central vacuole.The functions of the central vacuole include stockpiling proteins or inorganic ions, depositing metabolic byproducts, storing pigments, and storing defensive compounds against herbivores. It also increases surface to volume ratio for the whole cell.Endomembrane SystemThe endomembrane system plays a key role in the synthesis (and hydrolysis) of macromolecules in the cell.The various components modify macromolecules for their various functions.Mitochondria, Chloroplasts, and PeroxisomesOther Membranous OrganellesMitochondria and chloroplasts are the main energy transformers of cellsPeroxisomes generate and degrade H2O2 in performing various metabolic functionsMitochondria and chloroplasts are the organelles that convert energy to forms that cells can use for work.Mitochondria are the sites of cellular respiration, generating ATP from the catabolism of sugars, fats, and other fuels in the presence of oxygen.Chloroplasts, found in plants and eukaryotic algae, are the sites of photosynthesis.They convert solar energy to chemical energy and synthesize new organic compounds from CO2 and H2O.Mitochondria and ChloroplastsMitochondria and chloroplasts are NOT part of the endomembrane system.Their proteins come primarily from free ribosomes in the cytosol and a few from their own ribosomes.Both organelles have small quantities of DNA that direct the synthesis of the polypeptides produced by these internal ribosomes.Mitochondria and chloroplasts grow and reproduce as semiautonomous organelles.Almost all eukaryotic cells have mitochondria.There may be one very large mitochondrion or hundreds to thousands of individual mitochondria.The number of mitochondria is correlated with aerobic metabolic activity.A typical mitochondrion is 1-10 microns long.Mitochondria are quite dynamic: moving, changing shape, and dividing.Mitochondria have a smooth outer membrane and a highly folded inner membrane, the cristae.This creates a fluid-filled space between them.The cristae (folds) present ample surface area for the enzymes that synthesize ATP.The inner membrane encloses the mitochondrial matrix, a fluid-filled space with DNA, ribosomes, and enzymes. The chloroplast is one of several members of a generalized class of plant structures called plastids.Amyloplasts store starch in roots and tubers.Chromoplasts store pigments for fruits and flowers.The chloroplast produces sugar via photosynthesis.Chloroplasts gain their color from high levels of the green pigment chlorophyll.Chloroplasts measure about 2 microns x 5 microns and are found in leaves and other green structures of plants and in eukaryotic algae. The processes in the chloroplast are separated from the cytosol by two membranes.Inside the innermost membrane is a fluid-filled space, the stroma, in which float membranous sacs, the thylakoids.The stroma contains DNA, ribosomes, and enzymes for part of photosynthesis.The thylakoids, flattened sacs, are stacked into grana and are critical for converting light to chemical energy. Like mitochondria, chloroplasts are dynamic structures. Their shape is plastic and they can reproduce themselves by pinching in two.Mitochondria and chloroplasts are mobile and move around the cell along tracks in the cytoskeleton. Peroxisomes contain enzymes that transfer hydrogen from various substrates to oxygenAn intermediate product of this process is hydrogen peroxide (H2O2), a poison, but the peroxisome has another enzyme that converts H2O2 to water.Some peroxisomes break fatty acids down to smaller molecules that are transported to mitochondria for fuel.Others detoxify alcohol and other harmful compounds.Specialized peroxisomes, glyoxysomes, convert the fatty acids in seeds to sugars, an easier energy and carbon source to transport. PeroxisomesPeroxisomes are bounded by a single membrane.They form NOT from the endomembrane system, but by incorporation of proteins and lipids from the cytosol.They split in two when they reach a certain size. The CytoskeletonThe cytoskeleton is a network of fibers extending throughout the cytoplasm.The cytoskeleton organizes the structures and activities of the cell.IntroductionThe cytoskeleton provides mechanical support and maintains shape of the cell.The fibers act like a geodesic dome to stabilize a balance between opposing forces.The cytoskeleton provides anchorage for many organelles and cytosolic enzymes.The cytoskeleton is dynamic, dismantling in one part and reassembling in another to change cell shape.Other Cytoskeleton FunctionsThe cytoskeleton also plays a major role in cell motility.This involves both changes in cell location and limited movements of parts of the cell.The cytoskeleton interacts with motor proteins.In cilia and flagella motor proteins pull components of the cytoskeleton past each other. This is also true in muscle cells.Motor molecules also carry vesicles or organelles to various destinations along “monorails’ provided by the cytoskeleton.Interactions of motor proteins and the cytoskeleton circulate materials within a cell via streaming.Recently, evidence is accumulating that the cytoskeleton may transmit mechanical signals that rearrange the nucleoli and other structures. There are three main types of fibers in the cytoskeleton:MicrotubulesMicrofilamentsintermediate filamentsMicrotubules, the thickest fibers, are hollow rods about 25 microns in diameter.Microtubule fibers are constructed of the globular protein, tubulin, and they grow or shrink as more tubulin molecules are added or removed.They move chromosomes during cell division. Another function is as tracks that guide motor proteins carrying organelles to their destination.In many cells, microtubules grow out from a centrosome near the nucleus.These microtubules resist compression to the cell.In animal cells, the centrosome has a pair of centrioles, each with nine triplets of microtubules arranged in a ring.During cell division, the centrioles replicate.Microtubules are the central structural supports in cilia and flagella.Both can move unicellular and small multicellular organisms by propelling water past the organism.If cilia and flagella are anchored in a large structure, they move fluid over a surface.For example, cilia sweep mucus carrying trapped debris from the lungs.Cilia usually occur in large numbers on the cell surface.They are about 0.25 microns in diameter and 2-20 microns long.There are usually just one or a few flagella per cell.Flagella are the same width as cilia, but 10-200 microns long.A flagellum has an undulatory movement.Force is generated parallel to the flagellum’s axis.Cilia move more like oars with alternating power and recovery strokes.They generate force perpendicular to the cilia’s axis.In spite of their differences, both cilia and flagella have the same ultrastructure.Both have a core of microtubules sheathed by the plasma membrane.Nine doublets of microtubules arranged around a pair at the center, the “9 + 2” pattern.Flexible “wheels” of proteins connect outer doublets to each other and to the core.The outer doublets are also connected by motor proteins.The cilium or flagellum is anchored in the cell by a basal body, whose structure is identical to a centriole.The bending of cilia and flagella is driven by the arms of a motor protein, dynein.Addition to dynein of a phosphate group from ATP and its removal causes conformation changes in the protein.Dynein arms alternately grab, move, and release the outer microtubules.Protein cross-links limit sliding and the force is expressed as bending.Microfilaments, the thinnest class of the cytoskeletal fibers, are solid rods of the globular protein actin.An actin microfilament consists of a twisted double chain of actin subunits.Microfilaments are designed to resist tension.With other proteins, they form a three-dimensional network just inside the plasma membrane. The shape of the microvilli in this intestinal cell are supported by microfilaments, anchored to a network of intermediate filaments.In muscle cells, thousands of actin filaments are arranged parallel to one another.Thicker filaments composed of a motor protein, myosin, interdigitate with the thinner actin fibers.Myosin molecules walk along the actin filament, pulling stacks of actin fibers together and shortening the cell.In other cells, these actin-myosin aggregates are less organized but still cause localized contraction.A contracting belt of microfilaments divides the cytoplasm of animal cells during cell division.Localized contraction also drives amoeboid movement.Pseudopodia, cellular extensions, extend and contract through the reversible assembly and contraction of actin subunits into microfilaments.In plant cells (and others), actin-myosin interactions and sol-gel transformations drive cytoplasmic streaming.This creates a circular flow of cytoplasm in the cell.This speeds the distribution of materials within the cell.Intermediate filaments, intermediate in size at 8 - 12 nanometers, are specialized for bearing tension.Intermediate filaments are built from a diverse class of subunits from a family of proteins called keratins.Intermediate filaments are more permanent fixtures of the cytoskeleton than are the other two classes.They reinforce cell shape and fix organelle location.Cell Surfaces and JunctionsThe cell wall, found in prokaryotes, fungi, and some protists, has multiple functions.In plants, the cell wall protects the cell, maintains its shape, and prevents excessive uptake of water.It also supports the plant against the force of gravity.The thickness and chemical composition of cell walls differs from species to species and among cell types.Cell WallsThe basic design consists of microfibrils of cellulose embedded in a matrix of proteins and other polysaccharides.This is like steel-reinforced concrete or fiberglass.A mature cell wall consists of a primary cell wall, a middle lamella with sticky polysaccharides that holds cell together, and layers of secondary cell wall.Lacking cell walls, animals cells do have an elaborate extracellular matrix (ECM).The primary constituents of the extracellular matrix are glycoproteins, especially collagen fibers, embedded in a network of proteoglycans.In many cells, fibronectins in the ECM connect to integrins, intrinsic membrane proteins.The integrins connect the ECM to the cytoskeleton.Extracellular Matrix (ECM) The interconnections from the ECM to the cytoskeleton via the fibronectin-integrin link permit the interaction of changes inside and outside the cell. The ECM can regulate cell behavior.Embryonic cells migrate along specific pathways by matching the orientation of their microfilaments to the “grain” of fibers in the extracellular matrix.The extracellular matrix can influence the activity of genes in the nucleus via a combination of chemical and mechanical signaling pathways.This may coordinate all the cells within a tissue. Neighboring cells in tissues, organs, or organ systems often adhere, interact, and communicate through direct physical contact.Plant cells are perforated with plasmodesmata, channels allowing cysotol to pass between cells.Intercellular JunctionsAnimal have 3 main types of intercellular links: tight junctions, desmosomes, and gap junctions.In tight junctions, membranes of adjacent cells are fused, forming continuous belts around cells.This prevents leakage of extracellular fluid. Desmosomes (or anchoring junctions) fasten cells together into strong sheets, much like rivets.Intermediate filaments of keratin reinforce desmosomes. Gap junctions (or communicating junctions) provide cytoplasmic channels between adjacent cells.Special membrane proteins surround these pores.Salt ions, sugar, amino acids, and other small molecules can pass.In embryos, gap junctions facilitate chemical communication during development.While the cell has many structures that have specific functions, they must work together.For example, macrophages use actin filaments to move and extend pseudopodia, capturing their prey, bacteria.Food vacuoles are digested by lysosomes, a product of the endomembrane system of ER and Golgi.The enzymes of the lysosomes and proteins of the cytoskeleton are synthesized at the ribosomes.The information for these proteins comes from genetic messages sent by DNA in the nucleus.All of these processes require energy in the form of ATP, supplied by the mitochondria.A cell is a living unit greater than the sum of its parts.
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