Cell Biology: Understanding the Fundamentals of Life, Summaries of Biology

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G. J. Tortora, B. H. Derrickson,

Biology and physiology

Lecture 1 Introduction

• What is Cell Biology? o Fundamentals of cell biology: ▪ Chemical components and reactions and that govern: - cell structure - nutrient/energy assimilation ▪ Macromolecular assemblies and interactions that enable cells to perform such life-supporting tasks as: - Storage, processing, propagation of genetic info - Molecular transport - Communication with the external environment
• Common threads among more than 10 million species o iGrowth and reproduction o Communication with the environment o Acquisition and assimilation of energy o Homeostasis
• Cell theory o Cells are the functional units of life o All living organisms are composed of cells
• 8 common features of all cells o Cells are highly complex and organized o Cells possess a genetic program o Cells are capable of producing more of themselves o Cells are biochemical factories that constantly acquire and utilize energy o Cells engage in mechanical activities o Cells are able to respond to stimuli o Cells are capable of self regulation o Evolution first happens at the level of molecules and cells
• Model eukaryotes o “model organisms” have been chosen in which ▪ The genome has been sequenced ▪ Site-directed and tissue-specific mutagenesis is possible ▪ Expression of multiple genes can be tracked across many cells simultaneously ▪ The developmental sequence is known ▪ Examples: yeast (a minimal eukaryote), Arabidopsis (Common Thale Cress), C. elegans ( a nematode worm), Drosophila melanogaster (fruitfly), mouse Lecture 2 Visualizing Cells
• Light Microscopy o Limited in resolution (ability to distinguish 2 objects that are close to each other) ▪ Practical limit of resolution in light microscopy ~0.2 μm o Typical animal cell is 10-20 μm in diameter, mitochondria (~0.5 μm) are generally the smallest organelles that can be clearly seen my light microscopy

▪ Uses laser light that is reflected off the cover slip ▪ Molecules close to the cover slip (within 100-200 nm) can be excited by the EM field, providing a very narrow band of fluorescence o 2 - photon microscopy ( vital microscopy ) ▪ Uses 2 separate long-wave photons of light, instead of one short-wave photon, to excite the fluorophore ▪ The longer the wavelength of light, the further into the sample it can penetrate ▪ Allows much deeper penetration into a sample without sectioning → can use living organisms

• Electron microscopy o A focused beam of electrons replaces light. Resolution more than 200x that of light microscopy can be achieved ▪ Energy and wavelength are inversely proportional (higher energy = shorter wavelength); electron have more energy than photons and thus shorter wavelengths ▪ The shorter the wavelength, the better the resolution o Vacuum is required, so special techniques are required ▪ Electrons are matter, so they will interact with other matter like air. If we shined a beam of electrons through a column of air, the electrons would bounce off of the other atoms in air because the atoms have electrons and electrons repel each other and be scattered o Cells must be fixed (glutaraldehyde, osmium tetroxide) , and often desiccated (dried), and then sliced into ultra-thin sections no live microscopy o Cells are mostly transparent to electrons, so electron-dense materials are used to stain cells ▪ Can also use gold-tagged antibodies to mark specific proteins/structures o Scanning electron microscopy (SEM) images the outside surface (staining, but no sectioning) o Transmission electron microscopy (TEM) images internal structures
• An electron beam gives more resolution than light because the wavelength of an electron is much smaller than the wavelength of a photon
• Flow Cytometry o Live image visualization ▪ Live cells are analyzed in real-time in an aqueous stream as they pass through a laser o Computer can collect fluorescence data (color, intensity), as well as laser scatter (reflecting size, shape, and internal architecture) o Can be coupled with a cell sorter to allow cells to be separated based on fluorescence and scatter properties Lecture 3 Biological Molecules
• Building blocks of the cell → larger units of the cell o Sugars - > polysaccharides o Fatty acids → fats, lipids, membranes

o Amino acids → proteins o Nucleotides → nucleic acids

• Monomers are linked together to form polymers via condensation reactions o Specifically, dehydration reactions (because a water molecule is formed in the process) o Monosaccharides form glycosidic bonds, proteins form peptide bonds, nucleic acids form phosphodiester bonds
• Carbohydrates o Energy source, structural support, binding surface o Chemical features: linear chain <-> ring, several OH groups o Chemical nature: highly polar
• Describing sugar linkages o Carbons in the ring are numbered clockwise from the oxygen in the ring o The positions of the OH groups attached to each carbon in the ring are described as being either: ▪ UP (above the plane of the ring) = β ▪ DOWN (below the plane of the ring) = α o A given sugar molecule has multiple OH groups located at various positions throughout the structure, so linked sugars are capable of forming a vast array of branched polysaccharide structures
• Starch and cellulose are both polymers made up of glucose subunits. We can digest starch but not cellulose because we have enzymes that digest the α linkages of starch, but not the β linkages of cellulose
• Lipids o Hydrophobic membrane barriers, energy source o Chemical features: hydrocarbon tails with polar COOH at one end o Chemical nature: amphipathic o Phospholipids: hydrophilic (polar head group + phosphate group + glycerol) + hydrophobic fatty acid tails
• Amino acids o Building blocks of proteins, can be metabolized for energy o Chemical features: uniform chemical structure with directionality (amino terminus – carboxyl terminus) and side (R) group variability ▪ Amino group + carboxyl group + side chain

that allows them to interact with polar head groups, but there is also more space between the polar heads than between the nonpolar tails, so steroids can push their way past the polar head group o Glucose diffuses very poorly/not at all through membranes o Water (very polar) is very poor in moving through plasma membranes ▪ Polar molecules do not readily cross a nonpolar barrier o Steroid hormones are largely nonpolar with a little bit of polarity that allows them to interact with the outer surface of the membrane

• Phospholipids and membranes o Most abundant lipids in plasma membranes are phospholipids o Phospholipids are amphiphilic/amphipathic – they have a polar head group and two non-polar hydrocarbon tails o One tail is generally saturated (no double-bonds) while the other is unsaturated (one cis double-bond), which increases membrane fluidity
• Sterols and membranes o Another important lipid component of membranes are sterols o Contain rigid ring structures that stiffen portions of the phospholipids – essential for the structural integrity of the membrane o In animal cell membranes, the major sterol is cholesterol. Plants cells use a combination of phytosterols , and fungi use ergosterol
• Ergosterol and antifungal drugs o Because ergosterol is found in fungi, but not animal cells, it is a useful target for antifungal drugs o Amphotericin B and nystatin specifically bind ergosterol and form ion pores, allowing ions to leak out of the cell o Miconazole and Lamisil inhibit ergosterol synthesis, blocking fungal cell growth
• Self-assembly of phospholipid membranes o Due to their amphiphilic nature, phospholipids can self-assemble into lipid bilayer membranes o Polar head groups associate with water on the “outsides” of the membrane o Hydrocarbon tails associate with each other on the “inside” of the membrane
• Fluid mosaic model o The properties of the plasma membrane are described by the “fluid mosaic” model ▪ How is the plasma membrane a fluid? - The plasma membrane has the consistency of vegetable oil at body temperature → the proteins and other substances are able to move across it ▪ How is the plasma membrane a “mosaic”? - Proteins and substances such as cholesterol become embedded in the bilayer, giving the membrane the look of a mosaic
• Membrane asymmetry o Outer leaflet does not look like inner leaflet, important for cell’s functionality ▪ Sets up a charge differential across the membrane; inner leaflet is negatively charged, outer leaflet is neutral

▪ Molecules on outer leaflet can interact with other molecules in extracellular environment ▪ Molecules on inner leaflet can interact with other molecules in intracellular environment o Outer leaflet tends to be more enriched in sphingolipids and neutral phospholipids o Inner leaflet is rich in negatively charged phospholipids (phosphatidylserine)

• Membrane proteins o Plasma membrane contains proteins, as well as lipids o Provide many important functions ▪ Molecular transport ▪ Signal transduction ▪ Anchorage to the cytoskeleton ▪ Interaction with other cells/extracellular matrix o Proteins may be peripheral (bound to the membrane surface) or integral (inserted into the membrane interior) membrane proteins o Integral membrane proteins may span both leaflets via a transmembrane region , or may be anchored via attachment of lipid groups that insert into one leaflet Lecture 5 Nucleus and chromosomes
• Plant cell wall o Every plant cell is surrounded by a carbohydrate matrix called the plant cell wall o This is separate from, and outside of, the plasma membrane of the plant cell o The major component of the plant cell wall is cellulose , a polymer of glucose that provides tensile strength comparable to steel o Cellulose microfibrils are interwoven with pectin (a complex mixture of polysaccharides) that provides resistance to compression o Other components include additional cross-linking polysaccharides and lignin (waterproofing)
• Turgor pressure o The structural rigidity of the plant cell wall allows the generation of a large internal pressure: turgor pressure o When the intracellular environment has an excess of solutes, water will flow into the cell via osmosis o Without a cell wall, this would cause the cell to swell until equilibrium is reached, or until the cell burst o The cell wall provides resistance to swelling, even under hydrostatic pressure of 10+ atm o This provides rigidity to the cells (and is the reason plants wilt when dehydrated), and is also the driving force for cell expansion during growth
• Animal cells lack a cell wall. If they are placed in a highly hypotonic environment, they will expand until they burst
• The nucleus o Most obvious internal structure of eukaryotic cells and their defining characteristic o Contains the genetic material of DNA within a double membrane structure that is continuous with the ER

o The actual packing structure of the 30 nm fiber is not known, and several different models have been suggested, based on different imaging techniques o Interactions between the N-terminal tails of the histones in neighboring nucleosomes are important in forming the 30 nm fiber o The linker histone H1 binds to the outside of each nucleosome and also helps to condense the chromatin o 30nm fibers are still much too long to fit into a nucleus, so further condensation is needed o Mitotic chromosomes represent the most highly condensed form of chromatin o As with the lower levels of packing, the fine details are unknown, but can be postulated

• Heterochromatin and Euchromatin o Interphase chromatin is not homogenous, but consists of at least two types o Heterochromatin is highly condensed, and represents DNA that is resistant to gene expression (“silenced”). It also includes some specialized structures (centromeres, telomeres) o Euchromatin is less condensed and remains accessible to the RNA transcription machinery o Chromatin structure can be regulated by covalent modification to the histone tails
• A bit more on chromosomal structure o Within each chromosome are several specialized sequences that are required for proper duplication o The replication origin is a site where DNA duplication is initiated ▪ Eukaryotic chromosomes generally have many replication origins, to allow for more rapid duplication o The centromere is the site of attachment to the mitotic spindle, which allows one copy of each duplicated chromosome to be pulled into each daughter cell during mitosis o The telomere prevents the ends of the chromosome from being mistaken for broken DNA, and allows for proper duplication of the chromosome ends Lecture 6
• Endosymbiont theory o Mitochondrion contains its own ribosomes, evidence that it was once a free-living bacterium o Bacterium initially had single membrane, when bacterium was endocytosed by cell, it was surrounded by the cell’s plasma membrane ▪ Like the nucleus, the mitochondrion is surrounded by a double membrane structure. Unlike the nuclear structure, the two membranes are completely separate independent membranes, not a folded-over structure, that are different from each other ▪ Outer membrane looks similar to plasma membrane, has a lot of large channel proteins that allow material to move between cytoplasm and mitochondrion o Cardiolipins found in the inner membrane of the mitochondria can also be found in membranes of bacteria; this is a composition of the membrane seen in prokaryotes but in no other places in eukaryotes

• Mitochondria o Site of oxidative metabolism of carbohydrates, amino acids, and lipids o They generate most of the cell’s ATP: “powerhouses” of the cell o Double membrane structure ▪ Outer membrane: contains many porins (channel proteins) ▪ Inner membrane: rich in the special lipid cardiolipin; folded into cristae; site of the electron transport chain ▪ Intermembrane space: contains cytochrome c and several factors that regulate programmed cell death ▪ Matrix: site of oxidative metabolism; also contains mtDNA, ribosomes, and other components for expression of mitochondrial genome
• Mitochondrial DNA and the Search for “Eve” o Mitochondrial genome consists of multiple copies of a small circular DNA o Unlike the nuclear genome, the mitochondrial genome is inherited only from the mother o Using mtDNA from people around the world, researchers built an evolutionary tree based on the human maternal lineage o Result: convergence on a single woman in Africa 140-200,000 years ago, who is the ancestor of all current living humans
• Ribosomes o Protein-synthesizing organelles in all cells o Found in the cytoplasm and within mitochondria (and chloroplasts); not enclosed by membrane o Large complexes of protein plus RNA: over 80 proteins in eukaryotes (55 in prokaryotes), and 4 different RNA molecules (3 in prokaryotes) o Composed of large and small subunits, which only assemble when protein synthesis initiates o Often associated with endoplasmic reticulum
• Endoplasmic Reticulum o Complex membranous organelle that extends throughout the cytoplasm o Constitutes ~50% of membrane in typical eukaryotic cell o Continuous with nuclear membranes – ER lumen merges with nuclear intermembrane space o Divided into “rough” ER and “smooth” ER ▪ Rough ER is associated with many ribosomes, giving it a rough appearance in microscope image ▪ Smooth ER lacks attached ribosomes o Serves many functions in cells o Major storage site for calcium ions, which is important for cellular signaling o Smooth ER is the site of lipid synthesis, including sterols, and detoxification of lipid- soluble compounds o Rough ER is the site of synthesis of transmembrane and secreted proteins (plus proteins found in lumens of organelles along the secretory pathway)

o RNA transcription stops after RNA pol encounters a special DNA sequence ▪ Prokaryotes: sequence is called a terminator ▪ Eukaryotes: transcription ends after reaching a polyadenylation signal o Most eukaryotic RNA requires post-transcriptional processing before it can be functional

• Pre-mRNA Processing o For RNAs that will encode proteins, processing is required before they are considered mRNA o First modification occurs immediately after 5’ end of RNA exist polymerase ▪ Addition of 5’ cap marks RNA as mRNA-to-be o Most protein-coding genes contain intervening sequence ( introns ) that interrupt the actual coding sequences ( exons ) o Introns must be removed by process of RNA splicing ▪ Carried out by the spliceosome ▪ Made up of small nuclear ribonucleoproteins
  ▪ Directed by RNA sequences found at intron-exon boundaries o Spliceosome assembles on pre-mRNA while it is still being transcribed, but splicing process may be delayed o Splicing process is extremely flexible – a given transcript may have many possible splicing patterns o Once transcription is complete, the RNA 3’ end receives a poly-A tail ▪ 3’ end of original RNA is cleaved off, then a series of ~200 A’s are added by a poly-A polymerase ▪ Poly-A binding proteins bind to the tail – important for export from the nucleus and later protein synthesis
• mRNA Export o RNA synthesis and processing all occurs in the nucleus, but protein synthesis occurs in the cytosol o Only fully processed, mature mRNA is exported from the nucleus – depends on removal of some proteins and addition/retention of others o Mature mRNA binds to nuclear export receptor , which guides it through the nuclear pore complex into the cytosol
• What about “other” RNAs o mRNA represents only ~5% of cellular RNA o Up to 80% of cellular RNA id ribosomal RNA – makes up the structural and catalytic core of ribosomes o rRNA is synthesized by RNA pol III and RNA pol I o Other “non-coding” RNAs have functions in pre-mRNA splicing, ribosome assembly, protein synthesis, regulation of gene expression, telomere synthesis, etc.
• Protein translation o Once mature mRNA has been exported to the cytosol, it can be translated into protein by the ribosome o … o Amino acid is coupled to tRNA by aminoacyl-tRNA synthetase ▪ Amino acid is first “activated” by conjugation to AMP

▪ Amino acid is then transferred from AMP to tRNA ▪ Synthetase proofreads for accuracy o Protein synthesis occurs N-terminal → C-terminal o RNA message is decoded by the ribosome ▪ rRNAs make up structural and catalytic core: ribozyme o translation elongation is facilitated by elongation factors, which use GTPase activity to allow proofreading and speed up ribosome translocation o translation terminates when ribosome encounters a stop codon ▪ instead of tRNA, a release factor binds to the ribosome, causing the hydrolysis of the peptidyl tRNA, releasing the completed protein o ribosome disassociates into separate small and large subunits, releasing mRNA, release factor, and remaining tRNA

• protein translation occurs on polyribosomes o a single mRNA may have several ribosomes translating simultaneously – this is called a polyribosome/polysome