All living things are composed of cells. They are the minimal subunits of life. This may seem obvious, but far more is entailed than is suspected. Biologists still do not understand the nature of the organizing capacity of cells for function and replication, even though they have been thinking about it for decades (relate to Schleiden[1838], Schwann[1839] and Virchow [mid 1850s, omnis cellula e cellula], implications of Darwin [1859]-descent with modification.

Simple single cells-Monerans and Protista (some Fungi)

Monerans are prokaryotes, Protista and others are eukaryotes.

Complex organisms [communities of cells] ( Fungi, Plantae, Animalia) relate to intracellular parasites such as viruses, prions- have some properties of life [certainly the molecular components], but do not independently reproduce so are not considered living

The invention of the light microscope (as an outgrowth of the telescope and lenses of high quality [lentil-shaped]) provided the first opportunity to examine the "microcosm" and lead to the discovery of cells (Robert Hooke, A. Van Leeuenhook). Relate to spontaneous generation /vitalism (L. Pasteur,1860s).

Several types of Light Microscopes: bright field, phase, differential interference, polarizing, fluorescence (resolution around 200nm)

The Electron Microscope was invented in the 1930s-magnification and resolution limits were extended greatly. (Resolution less than 1nm). Two main types-TEM and SEM. TEM fine structure from thin sections. SEM surface features, a 3-D image

What are we able to ‘see’ using microscopes?

Cells and tissues (as a reflection of multicellular organization into special

structural and functional arrangements)

Subcellular Structures-organelles, macromolecular complexes

Cytoskeletons-MT,MF, IF and cell surface attachment sites

Products of cells ( ECM [collagen fibers], biominerals, other secretions)

[No organelles in prokaryotes, a nucleoid represents the nucleus-equivalent.]

Plasma Membrane-the enclosure, basically a lipid bilayer interpenetrated with proteins.

Nucleus- double enveloped structure central to the housing of chromatin, chromosomes

Cytoplasm-viscous fluid in the interior of a cell, transparent, mostly water and solutes.

Organelles- membrane (called internal membranes) enclosed structures in the cytoplasm-mitochondria, chloroplasts, vesicles, Golgi appartatus, lysosomes, peroxisomes, vacuoles.

Macromolecular structures- extended internal membrane of the ER (may be considered an organelle also), ribosomes, polyribosomes, glycogen storage structures,proteasomes.

Cytoskeletal elements- related to shape,motility-MT, MF. Related to structural stability-IF, several classes, such as keratin (skin), neurofilaments (axons), desmin (sarcomeres, muscle cells).

Nucleus-most prominent organelle, two concentric membranes (inner and outer) make up a nuclear envelope penetrated by nuclear pores. Contains DNA (as chromatin, organized in chromosomes). DNA is the genetic material. Diploid and haploid refers to the total number of chromosomes-for example we have 46 chromosomes which represent 23 homologous pairs (other than in males-XY). Somatic cells have 46 (diploid, two sets). Germ cells [egg and sperm] have 23 (haploid, one set) which recombine at fertiliztion to give 46.

Mitochondria-ancient origins as symbionts , eubacteria –like. Energy producers for aerobic eukaryotic cells-Krebs cycle and ETS to form ATP via oxidation of glucose [cellular respiration]. Contain their own DNA (circular), ribosomes, mRNAs, tRNAs.

Chloroplasts-ancient origins as symbionts, cyanobacteria-like. Perform the essential function in plants and algae of photosynthesis. They generate O2 and organic molecules and are the primary producers of food for animal life on earth.

Internal Membranes- organized into compartments with specialized functions-ER produces materials for export-2 types, rough and smooth, protein synthesis, membrane synthesis, respectively.

Golgi apparatus associated by vesicles ( vesicular traffic) with ER, glycosylation and targeting of proteins for secretion (exocytosis).

Lysosomes are specialized products of the Golgi apparatus filled with hydrolases used to breakdown many materials brought into the cell by endocytosis.

Cytosol-remove membranes and organelles and you are left with an aqueous solution of large and small molecules, the cytosol.

Cytoskeleton- polymers in the cytoplasm of cells (protein subunits that undergo polymerization and depolymerization, dynamic associations).

Actin-microfilaments.

Tubulin-microtubules in flagella, cilia and in the spindle apparatus used to separate chromosomes during mitosis and meiosis.

Keratin,vimentin,GFAP,NF and desmin-intermediate-sized filaments.

Shape, movement, internal highways (as in directing vesicluar traffic from ER to Golgi to PM for secretion). Cytoskeletal elements associated with a number of types of proteins known as crosslinkers, cappers, severing, bundling and motor molecules.

Unity and Diversity of Cells-

Prokaryote vs Eukaryote; Animal vs Plant vs Fungi vs Protista.

Meaningful differences-size, shape, combinations of attributes (for example, cell walls, presence of organelles) and rates of replication.

Size: Microns for bacteria, up to mm for animals. Bacteria are the smallest and simplest of cells (cocci, rods, spirals). Shape fits function in both pro- and eukaryotes. For example, nerve cells and long axons; cylinders with cilia as in a Paramecium, flagellated as in a human sperm, amoeboid as in a macrophage, spindle-shape muscle fibers and sarcomeres.

Other attributes: cell walls in plants and monerans ( though compositionally different from one another).

Rate of division: Bacteria divide in minutes, eukaryotes somewhat more slowly

(some exceptions; well-fed yeast, embryonic cells).

Meaningful similarities in chemical composition-fats, sugars, proteins, nucleic acids.

Meaningful similarities in genetic material (universal code)-DNA. Instructions for growth, development, repair, homeostasis and reproduction in the genes and their products.

Meaningful similarities in orgin of life. Descent (evolution) from a common ancestor (a prokaryote). Basis for change is for the most part mutations /errors in fidelity of DNA replication. These kinds of changes in genes or sequences regulating genes provide for selectible differences in individuals (for better or for worse)-part of generation of variation which is the basis for natural selection ( fitness and success in reproduction/offspring). Sexual reproduction also provides for the reshuffling of genes in gene pool (recombination during gametogenesis) that further builds variation reflected in the altered genetic composition of offspring.

Bacteria-eubacteria and archaebacteria-differences in gene organization, cell walls, environments. E.coli and number of genes-4,000/complexity of DNA simple.

H. sapiens has100,000 genes and 600 times the DNA with great complexity.

Giardia-intermediate stage of evolution to eukaryote? Nucleus, but no mitochondria, nor chloroplasts.

Brewer’s yeast (S. cerevisiae) –simple eukaryote. Fast growing and important commercially and historically. Great source for study of genetic and cellular aspects of basic eukaryotic life processes ( for example, the cell cycle).

Protozoans-single cell organisms with great complexity, diversity and fierceness-Didinium and the consumption of a Paramecium (see Fig 1-32)

A model plant- Arabidopsis thaliana-advantages and disadvantages.

Model animals- a fly (Drosophila melanogaster), a worm (Caenorhabditis elegans), a mouse (Mus musculus) and us humans (Homo sapiens). As we learn more about each of these species of plants and animals, we learn more about ourselves. (Is this a fair representation of organisms upon which to base the generalities of cell and molecular processes? Some say yes, some say no. Consider expansion of species studied)

We are still only beginning to understand embryonic development and the factors that bring about the differentiation the hundreds of kinds of specialized cells we find in our bodies (although based on four basic types- epithelial, connective, muscular, nervous –see Panel 1-3 mammalian differentiated cell types).

We are only starting to understand the regulation of genes, the fixity of their expression in different cell types, the reversibility of the differentiated state (regeneration, nuclear transplantation and cloning), and how functional assemblages of cells coordinate behavior (neurogenesis, neural function, instinct, learning) and how cells lose control of their cell cycles and become transformed cells potentially leading to cancer.

We are still only scratching the surface of how cellular and organismal development and homeostasis is related to the evolution of life on Earth and of our species in particular. It is with what we do know about these material and energetic processes of cells and the unicellular and multicellular organisms represented by them, and the molecules and compounds of which they are composed and through which they function, that this course concerns itself.

Practical- -GRE, MCAT preparation

-Cell and Molecular Biology is a nexus for information arising from, and useful for many levels of biology and chemistry-genetics, biochemistry,ecology and evolution, histology, physiology, anatomy, animal behavior.

-General knowledge (Scientific literacy) for making informed decisions in the technological world in which we live (bases for life, reproduction, disease, heritage, evolution, behavior)

This chapter is meant to be a quick review of chemistry ( bonds, carbon, associations with water ) with emphasis on organic molecules important in maintaining life.

Matter is made of elements, of which there are naturally 92. Only a relatively small number of types of elements are involved in living organisms (for example COHNSP in organic molecules or water (95+% of an organisms weight/mass), plus modest amounts of (ions) Na, Ca, Mg, K, and trace (but important) amounts of Fe, Zn, Cl, I, Cr, Cu, Se, Mn).

Smallest particle of an element is an atom – made from protons⁺,neutrons^o, electrons^-.

Atomic #= protons

Atomic wt=protons+neutron (variation in neutrons in an element=isotopes). Atoms linked together by bonds to form molecules.

Dalton and atomic weight (H=1); Avogadro’s number (6x10²³ ) and molarity, 1 proton (H)=1/6x10²³g.

Mole is M grams of a substance, where M is its relative molecular mass (molecular weight) and will contain 6x10²³ molecules of the substance. 1 M C=12g;

1M glucose=180 g; 1 M sodium chloride=58g.

Molar solution= 1M of a substance in 1 liter of solution. Think mM, uM also.

(Figs 2-2,6,7,8,9,10,11)

Stability of atoms is determined by electrons and their shells (orbitals). These are discrete energy states for electrons (2,8,8,8…) with islands of stability (unreactive chemically) when orbitals are completely filled ( He, Ne, Ar), and quite unstable

( reactive) when they not complete (H, O, C, N).

The tendancy is to either steal electrons from one atom ( Cl from Na ) to form ions (cations, anions), and form ionic bonds (electrostatic interactions) resulting in compounds called salts, OR to share electrons (C and C,H,O,S,P), thus forming molecules with covalent bonds.

Unequal sharing (distribution) of electrons around nuclei resulting from differences in attraction of electrons to those nuclei leads to polar covalent bonds (H and O in H2O). Single , double and triple bonds are possible.

Water is the most abundant substance (compound) in cells. Molecules and compounds can be hydrophilic, hydrophobic or both (amphipathic/amphoteric).

Hydrogen bonds- attraction of partially + charged hydrogen in polar molecule (in water or proteins, nucleic acids) to a partially negative charged atom (such as H:O in individual water molecules, or =O and HN in proteins [peptide bonds in different parts of polypeptide, 2⁰ structure], as well as bases in double helix of nucleic acids [A:T(U), G:C in DNA, RNA]).

van der Waals attraction –proximity effects; attraction and repulsion, based on fluctuating charge distributions around atoms.

Hydrophobic interactions- like favors like, a repulsion from water leads to associations (3^o structure of proteins, domains and folding)

Different bonds have different lengths and strengths ( energy needed to break a bond)

covalent @ .15nm and 90 kcal, ionic @0.25nm and 80 kcal in solids (3 kcal in water), hydrogen bonds@ 0.3 nm and 4 kcal (1 kcal in water), van der Waals @ 0.35 nm and 0.1 kcal

Dissociation of water or other polar molecules (acetic acid, hydrochloric acid) to release H+ to another molecule ( H₂0----H₃0⁺+OH^-). Substances that release H+ are acids (more H⁺=, more acidic).

Equilibrium state for water, H⁺ at 10^-7 M, pH scale is negative log of concentration, so pH=7 for water in equilibrium). Regulation/control of acidity in cells is very important.

The opposite of an acid is a base (a substance that accepts H⁺). Regulation of bases (strong OH-, weak NH₂) or alkalinity is also important for cells. Most Cellular fluids and extracellular fluids are maintained at or around neutrality, pH 7.2-7.4.

Molecules in Cells

A cell is formed from carbon compounds. Organic compounds arise from the versatility of C to form multiple bonds, rings, and polymers. Important chemical groups that occur repeatedly in organic compounds are:

bases (to form nucelosides, nucleotides)

are able to form multimers (oligomers, polymers such as polysaccharides, polypeptides, nucleic acids) or macromolecules (assemblages-phospholipids in bilayers form from hydrophobic interactions).

These molecules have roles in cell structure, function and energy sources (or all three.)

Carbohydrates/Sugars-monosaccharides (CH2O)_n n=1-7. Glucose, for example (C₆H₁₂O₆) and its set of isomers, optical isomers, D and L. D predominates.

Oligo and polyforms- several to hundreds, thousands or millions.

Examples-glycogen, starch, cellulose (glucose polymers), chitin (GluNac polymer)

Condensation reactions (1)-generating water to form multimers: anabolic

Hydrolysis reactions (2)–consume water to form smaller units: catabolic

For example, (1) glucose+glucose = maltose + water

Or (2) maltose+water= glucose+glucose

Fatty acids are components of cell membranes (general class-Lipids-which contain highly insoluble molecules-HC chains, isoprenes or sterol aromatic ring structures)

Hydrocarbon part and carboxyl group. Has hydrophobic and hydrophilic properties-called amphipathic.

Saturation-number of H on C-C backbone (C-H) bonds vs C=C bonds.

Triglycerides- (storage forms, great energy source, but also insulation and padding); glycerol OH linked via carboxyl group to fatty acid(s).

Phospholipids: principal in lipid bilayer making up plasma membrane and internal membranes of all types of cells.

A varied class of molecules containing a carboxyl group, an amine group attached to an alpha carbon, which itself has different functional groups covalently linked to it (R groups) . Capable of polymerization to form chains (COOH-NH2; peptide bond) with N-C polarity.

20 types of amino acids- nonpolar, uncharged polar, acidic, basic-used throughout living organisms in nature. Optical isomers-D, L . L forms in proteins (with some exceptions-bacterial cell walls, antibiotics).

The sequence of AA determines protein structure (i.e. , folding sensitivity, with variation based on local changes and weak interactions ); sequence of AA is determined by genetic code in sequence of RNA, derived from sequence in DNA.

Nucleotides are the subunits of DNA and RNA( which contain and transmit hereditary information)

Nucleotides made up of a nitrogen containing ring linked to a five carbon sugar

( ribose, deoxyribose) and it carries one or more phosphate groups. Nitrogen containing rings are bases ( they accept a H+ in acidic conditions).

Cytosine(C); Thymine(T) and Uracil(U) are pyrimidines (6 member pyrimidine ring.

Adenosine(A) and Guanine(G) are purines. ( 6 member ring fused with a 5 member ring)

In DNA ATCG are used, in RNA AUCG are used. (U sub for T)

DNA is double stranded-requiring hydrogen bonds (and hydrophobic interactions) to hold the chains together via base pairing (A-T, C-G, one pyrimidine with one purine). Chains are antiparallel.

RNA is single stranded (though capable of forming double stranded structure [even triple]). Phophodiester bonds form from reaction of nucleoside triphosphates (energy rich) at 3’OH of growing chain (all nucleic acids are synthesized 5’ to 3’) to make a chain of indeterminate length (depending on a number of variables, conditions). Sequences are read from 5’ to 3’.

Macromolecules contain a specific sequence of subunits.

Macromolecules are principle building blocks of cells. Structure-function relationships- change structure change function.

Scale differences make properties of macromolecules unpredictable from subunits- DNA and RNA transmit hereditary information; Proteins have shape and dynamics that determines:

Binding to other molecules for assembly of structural materials( such as collagen, or cytoskeleton),

Stabilization of other molecules ( histones in chromatin),

Insertion into and through lipid bilayers (channels and carriers),

Recognition and catalysis/anabolic reactions associated with antibodies or enzymes,

Energy transducing properties that allow for conformational changes that provide basis for function of molecular motors (flagella, cilia, muscle contraction)

Thus, we (as reproductive individuals in a population) can only be so stable with respect to genetic information and its protein outcomes (mutation, errors, recombination, alleles), lest we flirt with extinction from lack of suitable variation ( gene pool).

Conformations and conditions. Weak bonds or interactions ( hydrogen, ionic, van der Waals, hydrophobic) determine the fine adjustment of shape changes (conformations) which may be essential for macromolecular function.

The Main property of living things: Order

Maintaining order takes energy. In this case order is in the assembly of large molecules from small, or the ordered disassembly of larger molecules into small (anabolic and catabolic reactions). The idea here is that cells are tiny chemical factories performing thousands (even millions) of different reactions every second in a highly order way.

Need atoms and energy from nonliving matter.

Enzymes make these reactions possible by catalysing reactions. Enzyme reactions in cells are generally connected in series

A—B—C—D---E---F--G—H and interconnected in networks as in Fig 3-2.

a b c d e f g

The sum of catabolic and anabolic reactions defines a cells metabolism. ( The details of these reactions are covered in a course in Biochemistry, not to be dealt with here)

Biological order made possible by the release of heat energy from cells.

Order to disorder- 2^nd Law of Thermodynamics- in the universe (or any isolated system, the degree of disorder can only increase (Entropy).

( 1^st law states that energy can be converted from one form to another, but cannot be created or destroyed, Fig 3-7).

From the point of view of Probability –system(s) will change spontaneously toward arrangements that have the greater likelihood, thus more disorder (entropy).

Example: Heads and tails among 100 coins- 100 heads highly improbable; 50 heads/50 tails more likely, therefore more disordered with higher entropy. How can cells, which are extremely high order structures, even be possible?

Cells are not isolated systems, they draw and disperse there energy from and into the world (system) around them in a successful attempt to establish order. They use food (organic materials), photons, inorganics (as with sulfides, chemosynthetic). There is a price to establishing cellular order, the order of cells comes only with a greater disorder in the system around them, articularly in terms of unuseable heat, thus the 2^nd Law is not violated (Fig 3-6) .

All animals live off the energy stored in the chemical bonds of organic molecules. These molecules also provide structural building blocks (atoms) for cells. The idea here is of trophic levels-involving vast networks of producers and consumers (expand to plants as producers, animals as consumers, herbivores, omnivores,carnivores) - at the bottom of all major food chains are plants and the process of photosynthesis ( Figs 3-9, 3-10).

Plants are able to obtain all the atoms they need from inorganic sources-

Energy is derived from radiant energy of the sun-photons captured by a system of energy carriers associated with chlorophyll to drive metabolism.

The process of gradual oxidation ( as opposed to the instantaneous release from explosive burning) of organic material ( rich in C and H) to form the most stable form of C (CO₂) and of H (H₂0) is favorable. This process is called respiration (and is carried out by both animals and plants).

Photosynthesis and respiration are complementary processes (Fig 3-10), as is fungal and microbial degradation of organic materials to provide for recycling of atoms and molecules from organic to inorganic forms in the biosphere.

Multistep processes ( enzyme catalyzed) are involved in oxidation.

Oxidation-any reaction in which electrons are removed from an atom, with concommitant

Reduction-a reaction in which electrons are transferred to an atom ( Figure 3-11). Polar covalent bonds represent unequal distribution of electrons, thus one atom is oxidized and one reduced ( for example in H₂O, O is reduced because it partially gains electrons and each H is oxidized because they partially loss one.

When a molecule in a cell picks up an electron it often picks up a proton (H+) A + e^- +H⁺ = AH

Thus; Hydrogenation= reduction (gain of electron),

Dehydrogenation= oxidation (loss of electron).

Enzymes allow energy to be extracted from organic molecules through small steps that involve the progressive oxidation (and reduction) of those molecules.

An example is burning paper, which is a transformation of organic material to H₂0 and CO₂ and heat, never the reverse. In the process there is an increase in entropy and a loss of free energy (energy that can be harnessed to do work).

Spontaneous chemical reactions occur only in the direction that leads to a loss of free energy ( downhill, energetically favorable).

However, notice that your book does not blow up even though the most favorable energy state of C and H are in CO₂ and H₂O. Takes input energy to get such a reaction to occur (Activation Energy-Fig 3-12).

The energy hill is overcome by increase in kinetic energy to the system ( the match in the last example), but cells have no such mechanism, they must find a way to LOWER the activation energy so that reactions may proceed at modest to low temperatures. This is done using enzymes.(Figs 3-13 and 3-14)

Enzymes bind substrates ( molecules, atoms) and change the relationships between the molecules from random interactions ( kinetic hits) to interactions that are forced by (selective) binding at an active site to have appropriate proximity and proper orientation (as well as subsequent repositioning do to confomational change of the enzyme (induced fit model).

(with a negative delta G, reaction will go spontaneously)

delta G = delta G⁰ +0.616 ln [B]/[A]

when [B]/[A] = 1, delta G = deltaG⁰

at equilibrium ( between concentration and delta G⁰, delta G = 0

so [B]/[A] = e ^{–deltaG0/0.616} see table 3-1

Activated Carrier Molecules and Biosynthesis

Energy is stored in chemical bonds in molecules (activated carriers) from which it is easily exchanged, and which are able to move through the volume of the cell (Fig 3-23)

Principal activated carriers: ATP and NADH/NADPH

Coupled reactions (energetically favorable reactions are coupled to energetically unfavorable reactions that produce an activated carrier

(Illustrated in Fig 3-24)

ATP is the most widely used activated carrier molecule

Adenosine triphosphate(ATP) Figs 3-25 and 26 (ADP to ATP to ADP). An example is the transfer of PO₄ group from ATP to an amino acid such as serine (energy favorable, phosphoanhydride to phosphoester) .

Energy stored in ATP is often harnessed to join two molecules together.

A-H + B-OH + A-B + H₂O (unfavorable)

but

B-OH + ATP = BOPO₃ +ADP (favorable)

A-H + BOPO₃ = A-B + PO₄ (favorable)

Overall A-H +ATP + B-OH = A-B + ADP + PO₄

(see fig 3-27A and B) for glutamic acid to glutamine.

NADH and NADPH are important electron carriers

NAD+ (nicotinamide adenine dinucleotide)

Each of these is capable of picking up a proton and two electrons (H^-, an hydride ion) and being reduced.( Fig 3-28)

What about the difference between NADPH and NADH? Same in transfer, but different with respect to the types of enzymes with which these coenzymes operate. NADPH is associated with anabolic reactions

NADH is associated with catabolic reactions involved in production of ATP through the oxidation of food molecules (glucose, citric acid cycle, ETS)

In cells NAD+ is high relative to NADH ( so NAD+ acts as an oxidizing agent), whereas NADPH is high relative to NADP+ ( so NADPH acts as a reducing agent).

ATP, NAD+, NADPH, as well as

acetyl CoA – high energy link to acetyl group

carboxylated biotin - to carboxyl group

S-adenosylmethionine - to methyl group

Uridine diphosphate glucose - to glucose

Chapter 4- How Cells Obtain Energy from Food

(pages 107-111)

Cells require a constant supply of energy to generate and maintain the biological order that keeps them alive. This energy is derived from the chemical bond energy of food molecules (fuel for cells).

Sugars are major fuel molecules ( plants make them, plants and animals use them). Process by which sugar is broken down (oxidation) (Fig 4-1)

C₆H₁₂O₆ + O₂ = CO₂ + H₂O + energy

Note: activation energy in respiration vs burning and the storage of useful energy(also fats, proteins)

The energy of this breakdown is transferred to activated carrier molecules (ATP, NADH)

Animal cells make ATP in 2 ways- cytosolic, anaeobic (covered here)

Mitochondria, aerobic (Chapter 13)

Food Molecules are broken down in three stages to produce ATP

Emphasis on Figure 4-2

Proteins, Lipids and Polysaccharides broken down-part of digestion (stage 1)

( Think: our digestive system or a cell’s lysosomes)

Stage 2 –glycolysis starts in cytosol and final oxidation is completed in Mitochondrion

Stage 3 - oxidative breakdown of citric acid cycle occurs in the Mitochondrion, production of NADH, transfer of e^- to ETS and uses O₂ to produce H₂O and indirectly ATP.

Oxidative phosphorylation ( ADP—ATP) resulting from oxidative process. Process is quite efficient in capturing available chemical bond energy (glucose, fatty acids) in the oxidation process.

Glycolysis is a central ATP-producing Pathway. Emphasize Fig 4-3

Know the major ingredients, reactants and products

1) Energy input,

2) 6C to 2-3C,

3) Energy generation.

Go over the specific steps in Panel 4-1 to see that 2 ATPs start the process with 2 NADH, 4 ATPs and 2 pyruvates produced.

Chapter 13 Energy Generation in Mitochondria

(pages 407-418)

Energy production is central to life as we know it.

In the beginning there was no oxygen. Anaerobic reactions occurred and little energy efficiency was realized. Fermentation reactions are representative of these low efficiency reactions (alcohol, lactic acid).

A more efficient way evolved- transport of electrons along membranes- central to conversion of light energy to chemical energy in photosynthesis. This type of mechanism is also located in present day mitochondria in the inner membrane. Time scale (Fig 13-2).

1) Electrons (from oxidation of food) are tranferred along a series of electron carriers ( Electron Transport Chain, ETS) . This transfer releases energy to transport H⁺ across the membrane and, thus, generate an electrochemical proton gradient (see Fig 13-3). Fig 13-4 for proton pumping.

2) H⁺ flow back across the membrane through a protein complex called an

ATP synthase, which catalyzes the conversion of ADP to ATP.

Mitochondria and Oxidative Phosphorylation

Mitochondria are present in nearly all eucaryotic cells with most ATP produced therein. The mitochondria etabolize acetyl groups in the citric acid cycle and produce CO₂ and NADH. NADH donates electrons to ETS and thus oxidizes NADH to NAD+. Electrons pass to O₂ to form water. Energy associated with electrons along ETS is used to pump protons. Protons cross the inner membrane to drive ATP production ( ADP—ATP). This is known as oxidative phosphorylation.

(Fig 13-5)

Refer to Figure 13-8 for high energy NADH/NAD+ system dependent on catabolism of acetyl groups entering via acetyl CoA. (Figure 13-9)

NADH dehydrogenase complex;

cytochrome b-c1 complex and

cytochromoxidase complex) .

Active pumping of protons

1) generates a proton concentration gradient (therefore a pH gradient pH=8 in matrix, 7 in IMS)

2) generates a membrane potential across the IM

Thus, there is the formation of an electrochemical gradient that favors movement of protons back through the IM. This is the proton motive force.

Enter the ATP Synthase. (Figures 13-13,14 and 15).

Tthe synthase is an ancient device found in bacteria as well as in the mitochondria of animals, plants etc.

-F₀/F₁ subunits make up this large complex

Fo = transmembrane H+ carrier F1 = ATPase. Synthase can produce 100 ATP/sec. Needs approximately 3 protons for each ATP produced.

The Shape and Structure of Proteins

In terms of structure, they are 3-D constructs produced from amino acid building blocks and provide various complex structural assemblages combining proteins with other proteins, lipids and carbohydrates. For example, filaments, sheets and spheres.

In terms of function,the 3-D structure provides the correct shape and folding to establish the activities of enzymes, transport channels, pumps, motors, transcription factors and various carriers. (Panel 5-1)

Opener- ATP synthase ( Chapter 13 and ATP production from H+ movement).

Amino acid sequence (primary structure) Figs 5-1,2 peptide bond

Covalent bonds

Polypeptide backbone

Side groups- polar and nonpolar (Fig 5-3)

Polar A) acidic-glu, asp B) basic-lys, arg, his C) polar, noncharged-ser, tyr, asn, gln, thr

Nonpolar-gly,ala, leu, iso, phe, trp, cys, met, pro,val

Weak bonds- H, vdW, ionic, hydrophobic interactions

(figs 5-4,5)

Denature /renature Fig 5-7

Proteins come in a wide variety of complicated shapes ( we have yet to figure out [predict] 3-D shape from primary sequence.

Structural motifs-a , b and random coils

These structures result from consistent patterns of H bonding between N-H and =O of polypeptide backbone.

In an a helix, H bonds occur between every 4^th peptide bond in the helix

-C=O////H-N-

with a turn in the helix every 3.6 aa. Example ,a keratin

Helical chains can also interact to form coiled coils. This is based on sequence in which hydrophobic aa are presented asymmetrically along the axis of the coil. Interchain hydrophobic interactions are very stable for their exclusion of water- IFs and

collagen ( a triple helix) Figs 5-10, 11.

In b sheets, organization can be parallel or antiparallel depending on the random coils connecting these motifs. Example, silk fibroin, feather keratin.

Proteins have several levels of organization

1^o sequence-peptide bonds

2^o as we have just seen a and b structure based on H-bonds

3^o intrachain folding- disulfide, H, ionic, vdW and hydrophobic

4^o interchain interactions- disulf, H, ionic, vdW, hydrophobic

They may all be present in a protein. Which often sets up domains.

Figs 5-12,13

Protein Domains

Differentiated from structural motifs, modular units of independent self-folding, organizing sequence 50-350 aa .

Example-Fig 5-12 for CAP (catabolite activated protein). CAP has a DNA binding domain and a cAMP binding domain. When the large cAMP binding domain binds cAMP it undergoes a confomational change that changes the confomation of the small domain that enables it to bind to DNA.

Few of the many possible polypeptide chains will be useful.

The argument here is that order is imperative and imposed by the process of synthesizing the polypeptide in the first place.

Evolution has maintaind certain combinations of sequence, motifs, folding and positioning of active groups, with variations in intervening sequence to enhance the range of specificities. For example, the proteases elastase and chymotrypsin seen in Fig 5-14.

Larger Protein molecules often contain more than one polypeptide.

Define: binding site ( sites of interaction between proteins, or other molecules. Subunits ( monomers to dimers to trimers……) for identical or nonidentical subunits see dimer of CAP or tetramer of neuraminidase with identical subunits, or tetramer of hemoglobin with nonidentical subunits ( a and b globin)

(Figs 5-15,16)

Filaments( simple, interactive, identical subunits assemble to form helix of indefinite length. Inherent asymmetry of subunit shape as in actin.

Sheets or tubes-tubulin ( hetergeneous dimers, a and b , form protofilaments aligned and folded to form a tube. Polymerization and depolymerization.

Spheres-virus coat proteins, clathrin in coated pits.

There are also assemblies into massive arrays involving RNA and protein ( ribosomes) or DNA and protein ( chromatin, polymerase complex).

Proteins ( 2^o, 4^o) Nucleic acids ( intrachain RNA, interchain DNA)

This structure minimizes free energy.

Differentiate globular (spherical) from fibrous. (Fig 5-12)

Extracellular molecules are often stabilized by covalent cross linkages.

S-S bonds (disulfide bonds) between cys and cys. Fig 5-22

The reducing environment in the cytoplasm prevents these type of linkages so cys are reduced S-H H-S.

How Proteins Work

Proteins bind to other molecules

Binding- stick together

Specificity- selective association based on recognition of shape and charge distribution ( one protein will bind only to one or a few molecules).

Ligand- any substance bound ( ion, molecule large or small)

Binding site-ligand region of binding to a protein.

Binding between proteins is based on weak bonds or associations (interactions) so that such interactions can be fairly readily made or unmade. There is great strength in many weak bonds acting collectively, and the ability to act together depends on their presentation on complementary surfaces. Anything that interferes with that match up has tremendous influence on binding.

Thus, conformational changes that change interactive surfaces, may reduce or eliminate their binding. A binding site is a confomational outcome- a cavity or groove on the surface of a protein which brings about interactions that need not be sequential in nature. Fig 5-24.

The binding sites of antibodies are especially versatile

(fig 5-25).

Antibody-class of molecule of Ig superfamily

Antigen-ligand recognized by an antibody.

Binding strength is measured by equilibrium constant

(so different antibodies bind to their ligands with different strengths)

A+B=AB (on rate) Kon

AB= A+B (off rate) Koff

Kon[A][B] = Koff [AB] [ ]=concentration in moles

Kon = [AB] =K (equilibrium constant)

Koff [A] [B]

So antibody plus antigen-- Kon -- antigen-antibody complex

Koff

Eventually a steady state ( on=off) or equilibrium. If K is large, then binding is strong.

This binding strength can be converted to number of H bonds ( and other weak bonds that are so important in determining binding between proteins and their ligands).

1H bond=1 Kcal/mole=K change of 10 (see Fig 5-26)

Ligand binding is only the first step in their function

Lysozyme cuts polysaccharides of bacterial cell walls- bacteria ruptures under osmotic pressure.

Reaction type-hydrolysis ( adds H₂O to break sugar links

Energetically favorable ( severed polysaccharide chain at lower G than intact chain, more disorder) Figs 5-28,29

Reactants must attain a transition state (higher energy to overcome activation barrier). Collisions with H₂O unlikely to attain such an energy state, so no hydrolsysis.

Enter the enzyme, which lowers activation energy and promotes transition state.

1) proximity of water to covalent bond

2) orientation of bond and water

3) strain due to induced fit.

4) Interaction with charges or environment due to enzyme aa in active site, conformational changes.

V=Vmax.[S]

[S] + Km

(see hand out)

For example, retinal (vit A derivative) attached to rod protein rhodopsin alters protein conform starting cascade that leads to electric signalling eventually vision.

Heme in hemoglobin binds and unbinds O2 based on partial pressures and changes in pH

Biotin and carboxylation (biotin as a vitamin)

Ions (usually divalent-Ca, Mg, Zn) as in Zn in carboxypeptidase or in carbonic anhydrase, collagenase.

A----- B -----C see Fig 5-33 multiple feedback

X

Y

Z

Feedback inhibition=negative regulation

Competitive-at binding site (active site)

Noncompetitive-at other than active site

Positive regulation also occurs- the product of one pathway stimulates the activity of an enzyme in another pathway- for example, ADP concentration increase activates enzymes in glucose oxidative pathways to stimulate ATP production.

Allosteric means ‘other shapes’ Figs 5-34,35

An active site is influenced by binding of ‘regulatory molecule’ at nonactive site.

Regulatory site mediates conformational change that activates (positive allostery) or inactivates (negative allostery) the enzyme (usually an enzyme complex or multimeric construct).

Covalent alteration of protein ( at ser, thr, tyr)Protein kinases ( add PO₄) Protein phosphatases ( remove PO₄)

GTP binding proteins can undergo dramatic conformational changes.

GTP binding proteins very important in signal transduction. (Fig 5-37)

Self-limiting effects due to inherent tendancy (instability) to undergo hydrolysis GTP to GDP + Pi

(We will talk in more detail about G proteins and their role in 2^nd messanger activation and signal transduction as well as about protein factors involved in translation.

1. muscle movement –myosin

2. cell motility- myosin

3. organelle transport-kinesin, dynein

4.chromosome movement during mitosis-dynein

5. DNA synthesis- topoisomerase, helicase

Coupling an energy source to conformational change

ATP, GTP : ATP—ADP + Pi or GTP ----GDP + Pi

Tends to drive conformational change that results in unidirectional movement.

Again, hydrolysis of NTP( nucleotide triphosphates) drives unidirectional, repetitive cycles of conformational change.

Proteins working together with NTP energy sources produces multple binding sites to be used and for sequential reactions to take place, conformations to be altered and mechanical changes to take place.

Review section on tightly bound molecules (or ions) that add extra functions to proteins.

For example, hemoglobin utilizes iron ions to bind oxygen and conformation changes in retinal when subjected to certain wavelengths of light. Remember that many enzymes bind ions that assist in the function of active sites ( example, Zn in carboxypeptidase).

Be aware that feed back inhibition (Figs 5-32 and 5-33) regulate the catalytic activities of enzymes and tie them to the needs of the cell.

In fact, in allosteric enzymes there are two binding sites that interact cooperatively, one a regulatory site ( either positive or negative) and the other an active site involved in catalytic activity

( review Figs 5-34 and 35). A basis for understanding allostery that it is a change in the conformation of a protein (or group of proteins) that confers a change in the function of that protein (or proteins).

Conformational change can also be driven by phosphorylation ( see Fig 5-36). Know how this occurs generally, via protein kinases and protein phosphatases, and keep in mind that such phosphorylation changes may be positive ( induce activity) or negative ( inhibit activity).

In addition, be aware that binding and hydrolysis of nucleotide triphosphates ( GTP or ATP to GDP or ADP) can also induce changes in conformation that result in activation or inactivation of proteins

(see Fig 5-37).

Review what a motor protein is and what it can do in the context of protein machinery when subject to periodic or cyclic conformation changes (see Figs 5-39,40,41).

This chapter deals with the structure and function of DNA. Know what a gene is. Be able to define or explain the following –

Be able to explain DNA replication ( as seen in figs 6-17, 20, 21 and 22). The point here is to know and understand all the factors that allow DNA to be replicated including the DNA polymerases involved( a and d ), helicase, primase, topoisomerase, single stranded binding proteins, lead strand and lag strand activities( continuous and discontinuous synthesis), Okazaki fragments and directionality in synthesis ( remember 5’ to 3’ synthesis of DNA).

Also consider the accuracy of DNA synthesis. What allows it to be accurate to 1 part per billion? Proof reading ( DNA polymerase as a 3’-5’ nuclease) and replacement of inappropriate bases ( remember complementarity). This is shown is figure 6-18 and further expanded in figure 6-19 to show why 5’-3’ synthesis is a necessity.

With respect to DNA Repair ( specifically DNA Mismatch repair, starting on page 200), know what a mutation is and the consequences of it with respect to replication, transcription and translation

( think about sickle cell disease fig 6-23). The idea here is to understand how potential mutations are corrected before they are made permanent. What are the steps in excision repair? Using figures 6-26 and 30 as a guide consider nuclease, polymerase and ligase activity.

DNA in your cells is continually suffering damage of many kinds from many sources. Know the sources ( x-ray, uv, chemical) and the kinds ( depurination, deamination and thymine dimer formation- figs 6-27 and 28) of chemical changes that are involved. Also be able to explain the outcome with respect to DNA replication of the persistence of a change in a nucleotide ( substitution or deletion) in the new strand ( see Fig 6-29). Be able to explain how the repair machinery of a cell may recognize the new vs. the old strand ( see Fig 6-25)- nicks and methylation.

Getting from DNA to protein constitutes a general process called gene expression. This chapter deals with the transcription of DNA to RNA and the translation of RNA to protein (Figs 7-1 and 2). This is considered the ‘central dogma’ of biology.

Know the differences between DNA and RNA ( sugars, U for T, strandedness, length-see figs 7-3 and 4). DNA is used as a template for RNA polymerases ( types I,II, III). Know in general what types of RNA they synthesize-rRNA (I), mRNA(II), tRNA(III) respectively-with some variation (for example 5S rRNA is synthesized by RNA pol’ase III, see table 7-1). Know the products of those RNAs, for example mRNAs translate to protein but tRNAs and rRNAs do not.

Transcription of RNA takes place in the same direction as DNA (5’ to 3’) so the template strand of DNA gives rise to a 5’ to 3’ product (see Fig 7-7). In the simplest systems ( bacteria), RNA pol’ase attaches to the DNA at a promoter (with a sigma factor) and stops at termination sequences( see fig 7-9). Either strand of DNA can be used as a template (Fig 7-10). Eucaryotic RNA synthesis is not as simple.

Eucaryotic RNA undergoes processing in the nucleus and involves distinct processing steps before export of the mature molecules through nuclear pores to the cytoplasm. For messenger RNA, these processing steps include the capping of the 5’ end of the mRNA with 7-methylguanosine and addition to the 3’ end of the mRNA with a poly A tail. Note that there are differences between procaryotes and eucaryotes with respect to processing ( Fig 7-12). In addition, be aware that eucaryotic genes are interrupted by non-coding sequences called introns, which separate coding sequences known as exons.(see Fig 7-13 and 14).

The introns in a primary mRNA must be removed by splicing them out. This process involves a class of enzymes composed of both RNA and proteins known as snurps or small nuclear ribonucleoprotein particles ( which are rich in uracil). I want you to know the splicing process and the snurps involved ( as shown in Fig 7-16). Know where U1 and U2 bind, know what a lariat is and how it is formed, know the GU/AG rule for determining the intron-exon borders and the fate of the excised RNA.

How might alternative splicing be carried out (for example see fig 7-18)? Think about the problems of controlling such splices and how the products of splicing may be different from one another with respect to the proteins that they encode. Is this important? Why?

In going from RNA to protein there is a change in chemical alphabet ( nucleotide bases to amino acids) . Be able to use the genetic code to make a polypeptide (using fig 7-20). Consider in this process the start (AUG)and stop sequences( UGA,UAA,UAG) associated with specific codons (triplet code sequence). Be able to explain what redundancy is and how the wobble hypothesis helps understand it .

What is a reading frame?- be able to use the RNA sequence to show the outcome of changes in reading frame. Why is the reading frame important?

Know the structure of a transfer RNA and the relationship of the anticodon to the codon (Fig 7-22). Know about the aminoacyl-tRNA synthetases and what they do to associate tRNA with amino acids( Fig 7-23)

Know the composition of ribosomal subunits ( RNAs and proteins-fig 7-25) and the binding sites associated with the fully assembled ribosome ( A,P,E) and what their roles are in translation( Figs 7-26 and 27). Be able to explain the three phases of translation-initiation, elongation and termination ( Figs 7-27, 28 and 30). Know what peptidyl transferase is and what makes this enzyme so important. What do the stop codons recognize? Release factors.

What is a polyribosome? How is it involved in the rapid synthesis of a single type of protein? (see Fig 7-31). If the rate of protein synthesis is important, is the rate of degradation of that protein also important? What is the half-life of an RNA or a protein? Be able to explain how cells degrade the proteins they produce.

Know what proteases are and where they are found in the cell ( Proteasomes and lysosomes). Proteosomes recognize polypeptides that have been linked to ubiquitin ( a small protein). The proteasome is the trash can of the cell, trash can lid an all.( fig 7-32) Old, damaged or altered proteins are degraded and their amino acids recycled into pools available for recharging tRNAs, and so the cycle goes, build up and breakdown ( anabolism and catabolism) of cellular molecules under the guiding regulation of the products of the thousands of genes in the genome ( summarized in the gene expression diagram of figure 7-33).

The section on RNA and the origins of life will not be covered on the test.

How does the structure of a chromosome lend itself to regulated expression? We know that the information for ‘becoming’ is in the sequence of DNA contained in the genome, but how does this process occur? How are the genes appropriate for differentiation of cells selected for expression as a multicellular organism establishes its body plan, tissues and organs? Part of the answer to this regulation lies in the information covered in this chapter.

What is a chromosome? Single enormous DNA molecule with associated proteins, constituting what is called chromatin. What is the diploid number of chromosomes in humans? The haploid number? What is a karyotype( figures 8-2 and 3)? What does it show? What are the two extreme states of chromatin in a cell capable of division? Think interphase and extended DNA fibers and mitosis and highly condensed chromatin ( Fig 8-4). But also be aware that chromatin can be more or less condensed in interphase cells (heterochromatin and euchromatin) and that these states have profound effects on the ability of the DNA in them to be used in gene expression.

What specialized DNA sequences ensure that chromosomes replicate efficiently? Telomeres, centromeres and origins of replication( Fig 8-5). For telomeres know also what the problem of replicating the telomeres is and how it is solved. Telomerase and the extension of the lead strand to accommodate a primer that will allow the lag strand to be completed ( see Fig 8-6). For centromeres know that the kinetochore allows attachment to the microtubules of the spindle apparatus for separation of sister chromatids. For origins of replication know that they exist in large numbers throughout eucaryotic DNA and allow for rapid replication during S phase.

What is a nucleosome? The basic unit of chromatin structure. Know what one looks like, of what it is composed and how what information we have about nucleosomes was determined ( Figs 8-8, 9).

The beads on a string represent the winding of DNA ( 1.7 x) around an octamer of histones ( two each of H2A,H2B, H3 and H4). What are histone proteins? What biochemical characterisitics do they have that allows them to interact strongly with DNA? What is linker DNA in this context? What are the higher order structures found for chromatin and how are they thought to arise ( Fig 8-10)?

What is heterochromatin? Euchromatin? How do they influence gene expression. Be prepared to explain X-inactivation in mammals (Fig 8-12) and what it might mean. What is a Barr body ( inactivated X) and what is the Lyon hypothesis (first to suggest Barr body was an inactive X chromosome).

What is the relationship of hetero- or euchromatin to position effects on gene expression? Use examples in yeast and flies to explain this phenomenon ( Fig 8-13).

What is the structure and organization of the nucleus of a cell? Be able to recognize the nuclear envelope, nuclear pores, the region of the nuclear lamina, position of chromatin (heterochromatic and euchromatic), the nucleolus, microtubules, intermediate filaments, centrosome and endoplasmic reticulum (Fig 8-14).

How do we know that cells generally change the genes expressed without altering the nucleotide sequence of the DNA itself? Nuclear transplantation studies ( as outlined in Fig 8-16). Think cloning of Dolly and other animals( and plants).

What are the levels of regulation of genes in a cell? Start with transcriptional regulation, then RNA processing control, translation control and protein activation/inactivation ( fig 8-17). Also consider the regulation of export and import into the nucleus.

Transcription is controlled by gene regulatory proteins binding to regulatory sequences of DNA. How does this work? Think about molecular shape and weak interactions ( Fig 8-18). Protein and DNA interact along the major and minor grooves, wherein proteins can contact the bases. There are a number of special motifs ( DNA binding motifs, Fig 8-19). Alpha helix and beta sheet conformations are crucial. Multiple helices (homeodomain binding protein), helix-loop-helix, zinc fingers ( helix, beta sheet and zn ions) and leucine zippers ( hydrophobic interactions between proteins forming dimers) are examples of motifs which provide specificity and strength of binding between DNA and protein.

Know what a repressor is. Know what an activator is. Examples in the book are simple and straight forward procaryotic systems. The tryptophan operon is regulated by the level of trp in the cell (Fig 8-21). Know what a promoter is, an operator, start site for transcription. Using the CAP protein as an example, be able to explain gene activation. In this case a weak promoter-RNA pol’ase interaction is altered to a strong association by the binding of CAP, which is activated by the binding of cAMP. These changes in the trp repressor protein and CAP are allosteric changes in nature.

Initiation of eucaryotic genes transcription is more complex than in procaryotes. How?

1. The RNA pol’ases themselves ( only one in procaryotes, three in eucaryotes – see Table 8-1).

2. Eucaryotic RNA pol’ases require transcription factors ( general transcription factors).

3. Distance makes the heart grow fonder- transcription factors in eucaryotes can bind to sites very distant from the start site to influence gene expression and either repress (repressor) or activate (activators).

4. Transcription must deal with the structure of chromatin, nucleosomes, and other eu- or heterochomatic features of the chromosome.

Know how the general transcription factors(TF) work ( Fig 8-23). Pay particular attention to TFIID and the TATA box ( and the TBP see Fig 8-24). The assembly of the full set of TFs with the RNA pol’ase results in phosphorylation of the polymerase (by TF II H) and the beginning of transcription.

Expression of eucaryotic genes can be controlled by TFs working at a distance

( even thousands of nucleotide pairs away). DNA can bend and fold back over itself

( usually needs a minimum of 500 base pairs to fold back 180^o ). The idea for this is depicted in Fig 8-25 (also Fig 8-26), in which an activating protein in shown binding to a site called an enhancer ( DNA sequence), folding back along the length of the chromatin and binding at a second site with the transcription initiation complex .

Nucleosomes can interfere with transcription initiation if they include a promoter in their loops. How to overcome this? Move the nucleosome out of the way. One way to do this is with changes in the core histones, letting them loosen from interactions with the phosphate backbone by altering the charge on the lys and arg ( for example by acetylation). For heterochromatic regions the packing of DNA is so dense that transcription cannot be initiated at all ( see X-inactivation and position effects, for example).

Eucaryotic genes are regulated by combinations of proteins ( see Fig 8-27). In this combinatorial control system we see different groups of proteins( TFs) working together to determine the expression of a single gene ( Fig 8-28). This way the rate of transcription ( not just on or off) can be modulated in response to cellular needs. Likewise, the expression of different genes can be coordinated by a single protein ( Fig 8-29). How might this occur? If the activity of a controlling TF was linked to the initial binding of a hormone or other factor ( the glucocorticoid receptor protein and the steroid cortisol) which results in an allosteric change, then the receptor could in turn be bound to the DNA and work with other TFs to turn on transcription. In this way a single TF might ( and does) affect many different genes.

Ultimately the combination of TFs affecting gene expression can create cells of different types (i.e., expressing different sets of genes) ( Fig 8-31) . For example, muscle cells and Myo D (a special TF). But if Myo D is introduced into certain other cell types, it converts those cell types to muscle cell phenotypes. However, not all cells can be altered in this way, suggesting that the history of changes through which a cell has gone during its differentiation may or may not predispose that cell to responsiveness to Myo D. An important principle here is that differences in cell types are produced by differences in gene expression.

Stable patterns of gene expression can be transmitted to daughter cells. The idea here is that cells of one type give rise to like-type cells when they divide. A muscle cell does not become a liver cell. This cellular memory is determined by proteins in a cell that persist in the cytoplasm of daughter cells ( Fig 8-32 and 8-33) and have a positive feed back on their patterns of gene expression ( or lack thereof).

One of the most dramatic recent demonstrations of the influence of a single gene regulatory protein is that of the homeobox gene product Ey in drosophila , which controls eye development in flies (see Fig 8-34). In this case, the expression of this gene was made to occur in a region where otherwise a leg would develop. What happened was that under the influence of the Ey protein an entire new organ was created on the leg. This certainly takes to absurd limits the old adage to "watch where you step".

Chapter 11 Membrane Structure

Enclosing all living cells is a fatty barrier that serves both to protect the cell and to control the traffic of molecules and ions into and out of it. This barrier is a phospholipid bilayer with proteins embedded in it (Fig 11-4). The lipids provide an immiscible layer which prevents unregulated exchange between the outside and the inside of the cell. The proteins provide the function components of the membrane, where they may act as carriers and channels ( as well as in other roles) capable of selective movement or translocation of solutes across the membrane. The membrane surrounding a cell is called the plasma membrane, the membranes making up the interior of the cell ( in eukaryotes) are called internal membranes and include the ER, Golgi, vesicles, NE etc. The model developed to describe the relationship of lipids and proteins in cells is called the Fluid Mosaic Model and explains the membrane as a 2-d fluid.

The membrane phospholipids have special properties: hydrophobic tails, hydrophilic heads and are therefore amphipathic ( Figs 11-5, 11-6, 11-11). The three major types of membrane lipids are phospholipids, glycolipids (sphingolipids, cerebrosides) and sterols, specifically cholesterol. There are several different types of phospholipids and glycolipids and they are often asymmetrically distributed between the two layers of the bilayer, such that some are exposed to the cytosol and some are exposed only to non-cytosolic environments ( Fig 11-17). Asymmetry is imposed in the ER, where lipids are assembled at the cytosolic face of the bilayer and then transferred to the noncytosolic face using special transporters known as flippases. Rotational and lateral mobility in the plane of the membrane readily occur( Fig 11-15), but flip-flop between layers does not.

Fluidity is very important to cell function. Fluidity of membranes is affected by hydrocarbon chain length, saturation of hydrocarbon chains and cholesterol. Cholesterol mediates the fluidity of the plasma membrane by inserting between phospholipids and glycolipids (Fig 11-16) and preventing their interactions so as to lower the phase transition( freezing) point or to stabilize the phospholipids as the temperature rises and kinetic effects destabilize the lipids.

What can pass through a lipid bilayer? Not much. However, small molecules such as water, glycerol. CO2, O2 and ethanol can. What can’t? Refer to Fig 11-20.

Membrane proteins are the functional molecules of cellular membranes. They serve many functions including: transport, act as receptors, anchor the membrane, act as enzymes ( see Fig 11-21 and Table 11-1). Know how proteins associate with the lipid bilayer ( Fig 11-22)-remember amphipathic nature of membrane. Know how the alpha helical conformation is important in single and multipass proteins ( Figs 11-24,25). Know what a beta barrel is and how it can act as a channel for transport across a membrane (Fig 11-26). What is an integral membrane protein? A peripheral membrane protein? How are membrane proteins solubilized?( Fig 11-28). Why is this important to know? Know how bacteriorhodopsin associates with the membrane of Halobacterium halobium and how light is used to drive ion transport. What are the energy-related consequences of such activities? ( Fig 11-29).

The plasma membrane is reinforced by the cell cortex. Know how this works in red blood cells ( Fig 111-32) and what the consequences of alterations in this reinforcement mean to a cell. Include spectrin in your considerations.

What is the distribution of carbohydrate on the cell surface? (Fig 11-33) Know that there is an asymmetry with respect to sugars-they are all exposed on the surface and not on the cytosolic side of the plasma membrane. The sugar coating of a cell is called the glycocalyx and includes glycoproteins, glycolipids and proteoglycans. What are some of the functions of the sugars at the cell surface. Know what a lectin is. (Fig 11-34)

Be familiar with the Frye and Edidin experiment ( as depicted in Fig 11-35) and be able to interpret results under different temperature conditions. Know at least four ways in which cells can restrict the movement of membrane proteins ( Fig 11-36) and how different distributions into membrane domains on cells or between cells affects physiological function ( for example in Fig 11-37). This includes knowledge of tight junctions in the establishment of domains ( apical and basolateral) in intestinal epithelial cells.

The transport of all but a few solutes requires proteins embedded in the plasma membrane (figs 12-1 and12-3 for simple diagrams). Such transporters are highly selective ( Na vs K, glucose vs amino acids) . There are basically two classes of membrane proteins- carrier proteins and channel proteins ( Fig 12-2), know the difference. Most channel proteins are ion channels.

Think of three types of transport (see Fig 12-5) - ( 1. simple diffusion –direct passage of a molecule through the lipid bilayer, 2. Passive or facilitated-using protein carriers/channels but requiring no cell generated energy and

3. active -requiring cell produced energy directly or indirectly. Also think about the way in which molecules/ions move in a concentration gradient. They move down the gradient from higher to lower concentration(entropy), or uphill, against the concentration gradient( ATP, chemical energy). Cells need both, and they are often linked.

Ions are extremely important to the function and survival of cells, and in fact, ion concentrations inside a cell are very different from those outside ( Table 12-1). For animal cells this means more K inside than out , and more Na outside than in. Thus, there are ion gradients across the membrane, which may establish electrical potential or membrane potential (a difference in charges across the membrane) . The concentration gradient plus the electrical potential set up an electrochemical gradient which influences the movement of many types of solutes into or out of a cell (Fig 12-7).

Carrier proteins are required for the movement of almost all organic molecules across the cell membranes. What is the structure of a carrier protein ( Fig 12-4-remember bacteriorhodopsin?) . How does it work? Know how a hypothetical model of a passive glucose carrier functions-Fig 12-6. Remember that proteins undergo conformational changes during binding that allow for function in transport just as in enzymes function in catalysis.

Active transport moves solutes against their concentration gradients. There are basically three ways to do it ( Fig 12-8) ATP-driven pumps, Coupled transporters,and light driven pumps.

ATP Driven Pump- Know how the Na-K ATPase works (Figs 12-9 and 12-11). Combine your knowledge of protein-protein interactions, protein-membrane interactions, ligand-protein interactions and protein phosphorylation and dephosphorylation in controlling this process. What effect does ouabain have on the Na-K pump?

Coupled transporters- There are two types and both use the downhill movement of one solute along its concentration gradient to drive the uphill movement of another different solute against its concentration gradient ( see Fig 12-13). If the driving ion ( usually Na) and the coupled solute ( for example glucose) are both entering the cell this carrier is called a symport. If the driving ion and the coupled solute are moving in opposite directions ( one in, the other out) the carrier is called an antiport.( Fig 12-12). An example of an antiport is the Na-H exchanger , a device for controlling pH in the cytosol.

Na-K Pump helps maintain osmotic balance in animal cells. The problem for an animal cell is the movement of water ( osmosis) into it ( Figs 12-15,16). Since solute concentration influences water movement, and solutes( such as the predominant ion , Na) are always leaking across the membrane, the pumping of Na out of the cell tends to balance water movement to a state of equilibrium. Ca ions concentration is also kept very low in the cytosol. In addition to a concentration gradient of considerable energy, Ca is also an important second messenger, and its presence above about 10^-7 M is detrimental to cell control of metabolism, grow and gene expression. There is an active Ca pump, which is a Ca ATPase that work just like the NaK pump in keeping Ca low in the cytosol.Animal cells have different pumping characteristics than plants ( Fig 12-17)

How do ion channels work? How is membrane potential established? Two important properties distinguish ion channels from simple aqueous pores- ion selectivity and gating. Selectivity is determined by the structure and composition of amino acids in the proteins that perform as channels. Gating is a property of the proteins to open and close the aqueous pore allowing ions to pass through them( and down their concentration gradients- see Fig 12-18). Work on the activities of ion channels has been done using patch-clamp recording methods ( Fig12-20). Movement of ions across the membrane changes the voltage across the membrane, so induces dynamic alterations in membrane potential, which cells use to activate processes such as nerve impulses.

There are basically three types of gated channels- ligand gated channels ( as seen in Fig 12-18 for acetylcholine receptor), voltage gated channels and stress activated channels.( Fig 12-22). The proteins of voltage gated channels have a voltage sensitive domain that responds to changes in electrical potential by undergoing conformational changes that open or close channels. Stress activated channel proteins are sensitive to mechanical forces ( auditory hairs-Fig 12-23) that trigger opening of channels that elicit ion flow and membrane potential changes.

The membrane potential of cells is governed by permeability to specific ions. In the case of animal cells this ion is K ( Figs 12-25 and 26). Know how animal cells establish their resting membrane potential and what a K leak channel is and does (pages 392-393). What is the Nernst equation and how is it used (Fig 12-27)?

How are eukaryotic cells organized? They are composed of specialized compartments within the cytoplasm. Know the compartments described as membrane-bound organelles and some of their functions (Fig 14-2 and Table 14-1). Be able to explain how a nuclear membrane and ER might have evolved (Fig 14-3) and how mitochondria may have arisen from the engulfment of an aerobic

prokaryote( Fig 14-4).

How do proteins get transported into and out of different endomembrane compartments in animal cells?Know the three mechanisms: 1. cytosol to nucleus through nuclear pores 2. Cytosol to ER, mitochondria, chloroplasts, peroxisomes by protein translocators, 3) proteins through the ER, Golgi, vesicle system via transport vesicles.(Vesicular traffic).

Proteins are sorted by signal sequences, which direct a protein from one compartment to another.The general plan for the three mechanisms is seen in Figure 14-5 and signal sequences listed in Table 14-3. You should know at which region of a protein the signal sequences are generally found and how they function (including how they might be experimentally altered-Fig 14-6).

Nucleus: proteins( and RNA) enter or leave the nucleus via nuclear pores ( NP, Figs 14-7, 8,9) without unfolding. The nuclear pore is complex and large (100 proteins, 120Md). Proteins moving into nucleus have a nuclear localization signal( lys,arg rich). Nuclear import receptors help the transported protein bind to and be recognized by the NP. Transport is energy requiring(GTP) . Know the parts of the pore inside and out as well as the general structure of the nuclear envelope.

Mitochondria (Chloroplasts): proteins enter ( after unfolding) through translocators embedded in the inner and outer membranes (Fig 14-10). The signal sequence is recognized by a receptor, which diffuses laterally to the translocator. The protein is pulled through the membranes to the interior ( matrix in mitochondria) where it refolds to functional form. The signal sequence is cleaved off. Unfolding is accomplished by chaperones and used ATP hydrolysis as an energy source to unfold. Movement of proteins to other sites in a mitochondrion requires other signal sequences, revealed after the initial signal is cleaved.

Proteins enter the ER while being synthesized. For mitochondria the process is posttranslational, for the ER the process of translocation is cotranslational. All proteins are synthesized in the cytosol. Proteins destined for the ER ( and beyond) have a signal sequence at the N-terminal that is recognized ( as the protein is being synthesized) and bound to a Signal Recognition Particle ( RNA and protein), which is bound to an SRP receptor ( also called a docking protein)in the ER membrane ( Fig 14-13). Binding of the SRP induces a pause in translation that starts again as the ribosome is transferred from the docking protein to the translocational channel. Synthesis continues and for a soluble protein results in the inward movement of the protein to the lumen of the ER. A signal peptidase releases the protein, now soluble in the lumen (Fig 14-14). Start and stop transfer sequences allow integral membrane proteins to be inserted into the membrane ( Fig 14-15), thus forming single or multipass proteins.

Vesicular transport involves budding and fusion of membrane bound units from one membrane surface to another. Both soluble and membrane-bound proteins are transported in this way. There are secretory ( exocytic) pathways and endocytic pathways using this mechanism( Fig 14-17). Know what a coated vesicle is and how it arises from assembly of clathrin in conjunction with adaptins at the site of receptors bound with cargo. The parts and process are clearly depicted in Fig 14-19. Furthermore, know how vesicles may be targeted-v-SNARES, t-SNARES(Fig 14-20) and fused (fusion proteins, Fig 14-21) with their target membranes.

Secretion pathways involve ER-Golgi-vesicles-plasma membrane-release to extracellular space. This all starts in the ER. Modifications of proteins in ER include 1. N-linked glycosylation (Fig 14-22) at asn via transfer of dolichol bound oligosaccharides, and 2) formation of disulfide bonds. Many proteins destined to remain in the ER have a C terminal retention signal (KDEL) which allows them to be returned to the ER if they are transported out. Proteins are further modified in the Golgi ( glycosylation). The Golgi has a complex structure (see Fig 14-24) which you should know ( cis network, trans network, cis,medial and trans plates and cisternae). Know how constitutive and regulated secretion operate (Fig 14-25).

Endocytic pathways-know how the following work 1) pinocytosis, 2) phagocytosis and 3) receptor mediated endocytosis. Consider how endo- and exocytic processes are balanced so that there is no net gain in membrane surface on a cell. With respect to receptor mediated endocytosis, know how vesicles are formed with loaded receptors, the endosomal interactions (early and late) , recylcing of receptors (see Fig 14-30 for transcytosis and degradation), passage of cargo to lysosomes and delivery of materials for the building and maintenance of the cell (Fig 14-29

Lysosomes are the principal sites of intracellular digestion-know what a lysosome is, how it maintains an acid environment (Fig 14-31), how its enzymes are targeted to it ( mannose 6 phosphate and its receptor) and the three pathways of degradation (Fig 14-32-phagocytosis, endocytosis, autophagy).

How do eukaryotic cells communicate? They use molecules in their environments as cues. These cues come is a variety of types-soluble , insoluble, membrane bound ( as on another cell surface). For multicellular organisms this is particularly important because it allows cells to coordinate their actions, a ‘social structure’ of cells in tissues and organs.

General principles- the process of signal transduction-information converted from one form to another. There are signaling cells ( produce a signal molecule-such as a hormone or growth factor) and target cells ( which have receptors for the signals and respond to them) . Signals can act over short or long distances- Fig 15-3 gives a summary of the forms of cell signaling-paracrine(autocrine, local mediators), endocrine( hormones), neuronal(neurotransmitters), contact-dependent (membrane bound molecules). Each cell has a limited number of signals to which it can respond ( limited receptors). Signals binding to their receptors may have a multitude of effects ( movement, shape, gene expression, metabolic changes). The same signal may different effects on different cells types( (Fig 15-5). Different combinations of signals/receptors can bring about different cellular responses (Fig 15-6). Receptors relay signals via intracellular signaling pathways. There are intracellular signaling molecules whose actions may be to activate other molecules in a signaling cascade( Fig 15-7 for general pattern). The crucial features of this type of cascade are laid out on page 487.

Some signals can cross the plasma membrane directly ( see Fig 15-8 for comparison of cell surface receptors Vs intracellular receptors) – hydrophobic signals include steroids and thyroid hormones (thyroxine) Fig 15-9. The process works by binding the steroid to a receptor which undergoes a conformational change to allow passage through a nuclear pore and binding to regulatory sequences of DNA and thus affect gene expression, which ultimately changes the properties of the cell (fig 15-10). Nitric oxide( NO, derived from arg ) can also pass directly through the membrane and has direct and immediate effects (Fig 15-11). Its target is guanylate cyclase which is activated to form cGMP, a potent signaling molecule.

For cell surface receptors , there are 3 classes- 1) ion-channel linked receptors 2) G-protein linked receptors and 3) enzyme linked receptors (Fig 15-12 for models).

Ion channel linked receptors ( also transmitter-linked receptors) convert chemical signals to electrical signals, restricted for the most part to the neurons ( movement of ions ( Na, Ca, K, Cl) through these channels induce changes in voltage potential to open voltage gated ion channels needed in generation of an action potential or its inhibition).

Intracellular signaling cascades acts as a series of on-off molecular switches. Most switches are proteins undergoing activation by phosphorylation ( or phosphorylation cascades) or binding ( and subsequent hydrolysis ) of GTP ( though there are small signaling molecules such as cAMP, cGMP Ca ions). We have talked about these in previous chapters, but their activites are summarized in Fig 15-13.

G-protein linked receptors are the most prevalent cell surface receptors. A large family with hundreds of members. They are 7-pass membrane proteins.(Fig 15-14), and as a class they respond to a wide range of different signals. The process is straight forward – G protein linked receptors activate G proteins ( Figs 15-15, 16) which in turn active enzymes in the cell. G proteins are intracellular proteins, some of which have inherent GTP’ase activity. Three basic groups of G proteins-alpha, beta and gamma. Know how they function. G alpha in activation of adenylate cyclase and phospholipase C (Fig 15-16,18); G beta-gamma in regulation of ion channels ( Fig 15-17). Activation of adenylate cyclase gives rise to cAMP; activation of phospholipase C give rise to diacylglycerol(DAG) and inositol triphosphate(IP3). CAMP, DAG and IP3 are second messengers.

cAMP is deactivated by another constitutive enzyme known as cAMP phosphodiesterase-this is so the activities induced by cAMP can be controlled- see example of cholera toxin ( page 495). cAMP activates enzymes known as cAMP dependent protein kinase ( A-kinase, A= cAMP) . This enzyme phosphorylates a ser or thr on number of target proteins ( which may themselves be enzymes) and activates (or inactivates) them ( see Fig 15-22 for some effects of this signaling pathway).

What if the target for the activated G protein is phospholipase C? This signals the inositol phospholipid pathway ( Fig 15-23) and has the added effect of releasing Ca for the ER. G alpha-GTP activates phospholipase C which splits inositol phospholipid into inositol 1,4,5 triphosphate (IP3) and DAG. The IP3 diffuses to the ER where it binds to channel protein regulating Ca transport, so Ca is released into cytosol. DAG and Ca bind to and active another protein kinase , Protein kinase C (C-kinase, C=Ca). Ca release is of great importance in regulating cellular activity, hence its sequestering in the ER is critical. Why? Ca has a number of Ca binding proteins in the cytosol, which are activated by binding. One of them is calmodulin , which after binding Ca, can then activate a class of protein kinases known a CaM kinases ( Fig 15-25). Intracellular signaling is fast, sensitive and adaptable, as shown for photorecptor responses to light which are mediated by G-linked proteins ( transducins) in response to light activated rhodopsin (figs 15-26 and 27)

Enzyme Linked receptors mainly respond to growth factors, which are for the most part local mediators. These receptors are single pass proteins. The cytosolic region of these receptors has enzyme activities that are dependent on signal binding ( Fig 15-28). The largest class of such receptors is the receptor tyrosine kinases. In fact these receptors form dimers ( or multimers) in response to signals. Each enzyme domain on the monomer phosphorylates its partner. This activated complex then binds other intracellular signaling molecules which are activated as long as they are associated with the phosphorylated complex. How does this complex get turned off? Protein tyrosine phosphatases, which remove phosphates and deactivate the complex ( or the complex may be endocytosed and degraded in the lysosomal pathway).

The receptor tyrosine kinases activate a GTP binding signaling protein know as Ras. Ras is an adaptor molecule ( Fig 15-29) which interacts with receptor tyrosine kinases and in turn activates other proteins. Virtually al receptor tyrosine kinases are coupled to Ras ( which itself is tethered to the inner surface of the plasma membrane). Ras resembles the G alpha subunit of the G proteins. The Ras activated cascade results in the activation of DNA binding proteins that affect gene transcription (Fig 15-30). Problems in the underactivity of Ras ( no response to GF) or overactivity of Ras ( first observed in cancer cells where it is permanently on and so acts as an oncogene) .

Protein kinase networks integrate information to control complex cellular behavior ( Fig 15-31). Notice the overlap in final effects of the G-linked receptor pathways and those of the receptor tyrosine kinase pathways and Ras. This system is influenced by the crosstalk between the different pathways. 2% of our genes ( 1000) code for protein kinases, making them some of the most important proteins in the cells repertoire. Integration of signals may occur in several ways, two of which are shown in Fig 15-32, which deals with the integration of signals from two different receptors by either affecting a single protein kinase

( reflected in a double phosphorylation requirement) or phosphorylating separate monomers which can only form an active dimer when both are altered. It is the integration of information from many sources that allows cells to respond communicate appropriately with their environments, the consequences of which often determine whether they live or die.

The intricate network of protein filaments and tubules that extends throughout the cytoplasm of eukaryotic cells is called the cytoskeleton. There are basically three classes of proteins involved in constructing the cytoskeleton- those that assemble microtubules(MT) ( tubulin), those that assemble microfilaments(MF) ( actin) and those that assemble intermediate sized filaments ( intermediate filament proteins or IFs) (See Figure 16-2) . Each or these classes of proteins is also capable of disassembly, so, to differing degrees, the filaments or tubules formed are dynamic ( polymerize-depolymerize) in nature. In addition, each of the classes has a set of associated proteins that aid in linking the filaments/tubules to useful functions in the cell. For MT , they are the microtubules associated proteins(MAPs), for MF they are the actin binding proteins and for IFs they are the IF associated proteins.

Starting with IFs :

several types- keratins, vimentin and vimentin-related( desmin, glial fibrillary acidic proteins), neurofilaments and lamins ( part of the nuclear lamina and the most ancient of the types -see Fig 16-7). Approximately 10 nm diam. Strong and durable. No polarity. All have alpha helical central regions with globular ends( Fig 16-4). They assemble from monomers to coiled-coil dimers, to tetramers, octamers and finally to 32-mers, which is the mature filament( see 16-4 for details). IFs are particularly important in establishing structural linkages within cells that can be linked between cells( desmosomes and keratins in epidermal cells). A genetic problem in the assemble and interconnectedness of keratin IF in epidermis may be reflected in skin easily disrupted as in the case of Epidermolysis Bullosa simplex.

Microtubules:

Basically protein products for two genes- alpha tubulin and beta tubulin. Form heterodimers that self-assemble into hollow tubules ( 25nm diam) with polarized protofilaments ( Fig 16-9), that is tubules with a + and a – minus end. Most growth takes place at the + end. Also a gamma type tubulin , which is associated with centrosomal material (Fig 16-10). MT undergo assembly/disassembly behavior called dynamic instability( Fig 16-12). Be aware that GTP and hydrolysis to GDP at binding sites on tubulin dimers are involved in stability of MT.

Grow of MT occurs at + end, but under conditions in which GDP is associated with cap tubulins, there is a catastrophic collapse. The minus end is preserved in oligomeric form by protection in the centrosome. This region of organization of interphase MT is called the microtubule organizing center (MTOC). Colchicine inhibits polymerization, taxol stabilizes the tubule.

The motor proteins associated with MT are dynein and kinesin ( families of proteins). MTs are polarized structures and dynein ( an ATP ase) generally moves unidirectionally towards the minus end of a tubules, while kinesin ( also an ATPase) generally move towars the plus end. These motor molecules can transport vesicles or organelles ( cargo) or interlink several MTs to allow for a sliding tubule arrangement that allows movement and shape changes( Figs 16-14, 15, 16,17).

Cilia and Flagella use MT to provide cellular movements( Fig 16-23). Know the structure of the axoneme ( the 9 + 2 array of MT and other components-Fig 16-22). This includes the position of spokes, nexin, the plasma membrane as well as MT and Dynein (as discussed in class). Know what a basal body is.

Actin Filaments ( Microfilaments)

Actin filaments are constructed from actin protein monomers and require ATP to be bound to the protein for assembly of filaments to take place( Fig 16-26). MF are 5-7 nm in diam. And polarized in their organization ( that is they have a plus and a minus end). Most growth takes place at the + end. Filaments may well undergo treadmilling as part of their dynamic nature. Natural inhibitors of MF include cytochalasins ( which prevent assembly) and phalloidin( which prevents disassembly). There is a wide range of proteins that associate with actin ( Actin binding proteins) which mediate its function ( Fig 16-27). Know the classes of effects ( bundling, severing ,capping, sequestering [thymosin,profilin], motor, crosslinking, etc.).

Cell motility or movement depends upon actin filaments. Know what lamellipodia and filopodia are and what they do ( Figs 16-28,29 ). Be able to relate the MF, integrins and extracellular matrix to cell movement ( ‘crawling’) (Fig 16-31). Of special importance to movement of cells are the motor molecules known as myosins (Figs 16-32,33). Know myo I and myo II and how they operate in the sliding of actin filaments. Know how a sarcomere is organized (Figs 16-34,35,36) and know the components involved-actin, myosin II, troponin, tropomyosin( see Figs16-37 and 39) and the process by which calcium is released and utilized (T-tubules, sarcoplasmic reticulum, nerve input , troponin C ) and how ATP and its hydrolysis by myosin brings about contraction.

Cells reproduce in an orderly fashion, duplicating its contents and then dividing in two . This is called the Cell Cycle ( Figs 17-1,4). It is essential for cells of all living things to divide. The cell cycle occurs in steps called interphase (G1,S,G2) and m-phase(mitosis and cytokinesis). Interphase involves cell growth and DNA replication and M-phase involves chromosomal separation and formation of two daughter cells.

Mitosis depends on the activities of MT ( spindle apparatus) and cytokinesis depends on the activities of MF (contractile ring) [Fig 17-5].

Some organelles fragment during mitosis( nucleus, ER, Golgi), others are randomly ( and fairly evenly) distributed between daughter cells.

Steps in Mitosis

What happens in prophase? Formation of mitotic spindle( centrosome duplicated in interphase moves around nucleus) , condensation of chromosomes.

What happens in Prometaphase? Disintegration of the nuclear envelope( phosphorylation of nuclear lamins), spindle fibers connect to chromosomes. Know what polar MTs are and what a kinetochore and kinetochore MTs are (Figs 17-6,7,9,10).

What happens in metaphase? Balanced distribution of chromosomes along equator of cell

(metaphase plate).

What happens in anaphase? Separation of chromosomes ( anaphase A) and separation of spindle poles ( anaphase B) (Fig 17-13). Anaphase A involves dynein motors that pull chromosomes to poles. Anaphase B involves kinesin motors that push spindle apart ( with polar MT) and dynein with astral MT to pull poles outward.

What happens in telophase? Events in reverse-NE reforms (Fig 17-14) with dephosphorylation of lamins, chromosomes decondense, ER reforms, Golgi reforms, syntheses start( transcription, translation).

Cytokinesis

Cytokinesis begins with formation of a contractile ring, the activities of which are first noticed as a cleavage furrow at 90 degrees to the spindle axis (Figs 17-15,16). Contractile ring formed of MF and myosin motors.Actin filaments slide over one another while attached to integral membrane proteins, thus pulling the plasma membrane inward to form a furrow.

Meiosis

Know the differences between mitosis and meiosis as indicated in Fig 17-23. Be able to define what an homologous pair of chromosomes is and how such are dealt with in mitosis and meiosis( Fig 17-20). What does it mean to be diploid? Haploid? What is a bivalent? A synaptonemal complex? What is crossing over and when does it occur in meiosis ( Fig 17-21)? What is it called when homologues fail to separate during meiosis? Be able to name a disorder in humans associated with this phenomenon.

The molecular control systems that regulate the cell cycle act in a highly coordinated fashion to control the progress through the cycle- G1,S,G2, M-phase ( 18-1). The controls can be viewed as checkpoints that allow entrance to certain crucial phases of the cell cycle, for example entrance to S ( DNA replication)and entrance ( spindle assembly, chromosome condensation) and exit from M ( trigger anaphase and cytokinesis) ( Fig 18-2 and Fig 18-3 for the types of monitoring that a cell may carry out at a particular stage in the cell cycle ).

Cell cycle control is based on cyclically activated protein kinases. They are activated by cyclins and called cyclin-dependent kinases or Cdks ( Fig 18-4). Know how the Cdks and cyclins were discovered ( for example what is MPF?)( use Figs 18-6, 7,8 and my graphs in class to differentiate activity Vs concentration of kinases, cyclins).

The activity of Cdks is also regulated by phosphorylation of the kinase. Activating kinase and inactivating kinase are involved ( see Fig 18-9). There is also an activating phosphatase, which is itself further activated by the Cdk ( Fig 18-10) in a positive feed back mechanism. Different cyclin-Cdks trigger different steps in the cell cycle- this is presented clearly in Figs 18-11 and 18-12 for the initiation of M-phase (MPF) and for S phase( S-phase Cdk). Cyclins are rapidly degraded by a ubiquitinating system that is activated by the cyclin-Cdk complex, thus inactivating the complex and subjecting the ubiquitinated cyclins to proteosome enzymes. The accumulation of cyclins reaches a critical point (in conjunction with activating and inactivating kinases and phosphatases) where it supports activation of the Cdk, and thus, the many phophorylating activities affecting the cell entering prophase/prometaphase (chromosome condensation, lamin breakdown, spindle reorganization). This Cdk activity is also the downfall of cyclins, leading to the rapid degradation of these proteins and an end to kinase activity.

Know how the cell cycle can be halted in G1 by Cdk inhibitor proteins. In this case consider damage to DNA as triggering p53, which in turn activates transcription the p21 gene, whose product(p21) binds with the S phase cyclin-Cdk complex and inactivates it ( See Fig 18-13). Also know what it means to withdraw from the cell cycle ( Go). Decisions, decisions. A summary of the checkpoints is presented in Fig 18-15.

How is cell number controlled in multicellular organisms? Cell proliferation, cell survival, cell death. Cell proliferation depends on signals from other cells. Know how the retinoblastoma protein( Rb) works ( as depicted in Fig 18-16) and how it is linked to GFs, such as PDGF ( also know other growth factors and their targets-FGF, NGF, EGF, HGF, Erythropoietin). Be able to follow the effects of damage that lead to thrombosis and to the release of PDGF. Why are they important? Consider hepatocyte growth factor as well. What is being regulated by these factors?

What is cell senescence? What are survival factors? How are these phenomena related to programmed cell death ( apoptosis)( compare apoptosis to necrosis). How might survival factors determine life and death of cells? (see Figs 18-20, 22). What is the suicide machinery of the cell as described in Fig 18-22? How are the suicide proteases amplified to bring about the neat, clean cell death of cell undergoing apoptosis? Be able to provide an example of a ‘killer signal’. What kinds of thing happen internally and at the cell surface of cells programmed to die?

When control of cell proliferation is lost ( through mutation), cells set free to divide indefinitely are very dangerous to the body-leads to cancer. Two many players in normal control are proliferation genes( also called proto-oncogenes, which when alter may become oncogenes) and antiproliferation genes (tumor suppressor genes, which when altered fail to stop a cell from over proliferating-examples: p53, Rb) ( See Fig 18-23). There are many sorts of proto-oncogenes and tumor suppressor genes coding for different proteins that can effect transformation of cells to cancer cells. If you consider the many types of proteins involved in cell signaling pathways ( GFs, receptors for hormones, GFs, G-proteins, ras, enzymes involved in producing or releasing 2^nd messengers), alterations in their presence, timing, activity, gene regulation etc., are often associated with initiation and progression of cancers.

Functional associations of cells in multicellular organisms are called tissues. There are 4 basic types-epithelial tissues, connective tissues, nerve tissues and muscle. Tissues are composed of cells and of extracellular matrix (ECM) materials. Cells are held together by specialized junctional complexes. The activities of cells in tissues, and of tissues in the organs and organ systems in which they are found, are highly coordinated and cooperative( Fig 19-2). ( We will not cover the sections dealing with plants-pages 594 to top of 600)

Animal connective tissue consists largely of ECM and particularly important is the fibrous protein collagen.( Figs 19-9,10,12). Mammals have about 20 genes for different collagens. Types I-IV are the most prevalent and important. Collagen is synthesized in the ER, forms triple helical coiled coils ( a soluble form called procollagen)and is transported through the endomembrane system and secreted into the extracellular space where it is altered by enzymes (collagenases) into an insoluble form that assembles into fibrils and fibers of great strength. Cell types best known for producing collagen are fibroblasts ( of connective tissue in general-skin, tendons), osteoblasts ( bone forming cells) and chondroblasts( cartilage forming cells).

There are molecules in the ECM which interact with collagen, while at the same time interacting with cells. One such molecule is fibronectin ( Fig 19-14), another is laminin. These molecules have binding sites for collagen as well as for integrins. The integrins in turn are linked to the cytoskeleton of the cell. This molecular linkage ties the cell ( through the cytoskeleton) to the framework (collagen) of the EMC and thus supports stable tissue structure. Other important molecules of the ECM are polysaccharides, particularly glycosaminoglycans(GAGs) (Figs 19-15[know the repeat sugar dimer of hyaluronate] and 16) and proteoglycans. Proteoglycans are combination of polysaccharides and proteins (core and linker types)that are often immense in size and mass ( millions of Daltons). These materials show up in less dense connective tissues and serve a number of important purposes, including space-filling ( from hydration), lubrication, anti-compression, GF binding, promotion of cell migration and osmotic activity.

Epithelia are probably the most ancient of forms of cell associations in evolving multicellular organism. They are tight packed together and have specialized junctions holding them together. They also have special ECM materials associated with their basal surfaces ( the basal lamina- made up principally of collagen IV, laminin and proteoglycans) . Epithelia come in simple and stratified types and in epithelial cells have different defining morphologies- cuboidal, columnar and squamous ( Figs 19-17,18). There is an inherent asymmetry to epithelial cells ( Fig 19-20), because they have to function as barriers, absorptive surfaces and secretory cells. The apical-basolateral axis defines the direction in which molecules move into the body ( absorptive cells) and out onto surfaces ( mucus from Goblet cells in the gut, for example).

There are a number of kinds of junctions associated with epithelial cells, including tight junctions, adherens junctions, desmosomes, gap junctions and hemidesmosomes.( Fig 19-21)

Tight junctions provide the means to form barriers to molecular migration or diffusion( Fig 19-22). These junctions prevent materials from leaking between cells ( in or out) and prevent the mixing of integral membrane proteins along the plane of the membrane from apical to basolateral ( or vice versa).

Adherens junctions and desmosomes are formed through the interaction of cadherin molecules on adjoining cell surfaces ( a homophilic interaction)( Fig 19-23). These cell adhesion proteins require Ca to function and are connected to intracellular proteins that in turn connect them to the cytoskeleton. Adherens junctions are associated with actin filaments, (Fig 19-24 for adhesion belts)while desmosomes are associated with IF ( keratins in epithelial cells) (Fig 19-26). Hemidesmosomes are quite different than desmosomes , even though they share a similar name. Hemidesmosomes attach cells to the ECM

(particularly the basal lamina) through integrin proteins interacting with laminin. They , like desmosomes, are connected to keratin IF.

Gap junctions form tiny channels between cells. They are also called communicating junctions because molecules can migrate from one connected cell to its neighbor ( also electrical activity is possible as in the case of cardiac myocytes). The gap junction proteins are multipass proteins known as connexins and they form multimeric, intercellular complexes known as connexons ( see Fig 19-28).

There are over 200 visibly different cell types in the human body. They are associated together in different ways to establish the specialized tissues that function in the maintenance and renewal of the body. Contributing to this organization and maintenance are 1. Cell communication, 2. Selective cell-cell adhesion 3. Cell memory ( see page 614 for details and Fig 19-31). Different tissue are renewed at different rates. Know what a stem cell is and why it is important ( Fig 19-32 and Figs19-33, 34,36 for variations on the theme) Know the difference between differentiated and determined with respect to stem cells.

Revisit cancer. A single mutation may result in a cell that violates normal controls on proliferation, survival, and differentiation. Two important aspects of cancer cells 1. Grow out of control and 2 invade and displace other tissues. Know what a tumor is , and what it means to be benign or malignant ( invasive and metastatic). Review oncogenes ( and the idea of gain of function mutations) and tumor suppressor genes ( and the idea of loss of function mutations) ( Ch 18). Cancer arises from somatic cell mutations(as opposed to germline mutations). In general cancer requires an accumulation of mutations, a single mutation is not enough. It appears as if 5-6 mutational events must occur, and this can take years or decades to accumulate ( see Fig 19-38 for colon cancer progression, APC is another tumor suppressor gene- how would having only one copy be a real disadvantage for cells?)