Section 1.1

Galileo, 1614

reported fine structure of a fly cuticle

Cesi, 1624

described a lens for looking at small things

Hooke, 1665

writes about cella in cork, lens is 30x

Grew and Malpigi, 1666

report cells in plants, breakthrough making plants and animals appear more similar

Leeuwenhoek, 1673

called red blood cells and all animal cells ‘globules’, lens was 300x

Kaspar Wolff

plants and animals have similar subunits, growth is due to growth of new vesicles

Henri Dutrochet

cells are units of metabolic exchange, everything is made of cells

Francois Raspail

Omnis cellula e cellula, cells make up the entire organized world

Dumortier, 1832

saw cell division in Conferva aurea

Robert Brown, 1833

coins term nucleus for the dark spot in the cell

Cell theory

all organisms are comprised of one or more cells

cells are the basic unit of life

cells only arise from pre-existing cells

Schleiden and Schwann

Foundation for cell theory tenet 1: all organisms are comprised of one or more cells

Remak, propagated by Virchow

Foundation for Cell Theory tenet 3: cells only arise from pre-existing cells

Cell biology

combination of biochemistry, cytology, and genetics

Fourth ‘tenet’ of cell theory

cells vary in size, shape and function

Micrometers

10^-6 m, 1 millionth of a meter, used for organelles and cells

Nanometers

10^-9 m, 1000 nm = 1 micrometer, used for molecules and subcellular structures

prokaryotes

no true nucleus, some organization, no membrane

eukaryotes

true nucleus, endomembrane system

Domains of life relationships

Eubacteria and eukaryotes split 3.5 bya, archaea split from eukaryotes 1.8 bya

Archaea and eukaryotes relatedness

despite appearing to be more related to eubacteria, archaea split from eukaryotes, making the term prokaryotes out-dated

Endomembrane system

ER, lysosome, endosome, Golgi, transport vesicles

Debated - cell membrane

Autogenous origin

ancestral prokaryote experienced an infolding of the plasma membrane around the nucleiod

Endosymbiotic origin

eubacteria absorbed an archaea (2 cells to 1 cell), requires a lot of corollaries/side hypotheses

Endosymbiotic hypothesis (Marguilis, 1967)

theoretical origin of mitochondrial/chloroplast → ancestral cells acquired bacteria that became energy-producing organelles (proven by later scientists)

Evidence for endosymbiotic hypothesis

autonomously replicating DNA genome, RNA polymerase/ribosomes resemble eubacteria, mitochondria deviate from universal genetic code, divide by fission like bacteria

Inside-Out Origin, Baum

archaea formed a nuclear membrane around the nucleiod during the process of absorbing eubacteria for mitochondria, membrane was once environmental space

All domains can have . . .

intracellular compartments, including membrane-bound structures

Compartmentalization allows for? (six things)

• distinct ionic and pH environments

• proteins encoded by nuclear genes must be targeted

• distinct functions for organelles

• cell size is not diffusion limited, so cells can be bigger

• toxic metabolic intermediates are isolated

• competition among biochemical pathways is reduced

co-translationally

protein targeting occurs with protein formation

post-translationally

protein targeted after formation

Vaults

compartment made of ribonucleiproteins, associated with drug resistance in blood cells

Biomolecular condensates

shell and membrane-less organelles, liquid-liquid phase separation via multivalent protein interactions, associated with RNA and stress biology, contain ordered proteins

Organelles

intracellular compartment that is specialized for a function

Second law of thermodynamics

in the universe or a closed system, trajectory will always be towards greater entropy

Cells do not violate the second law of thermodynamics, how?

cells generate order, but are not isolated systems → biological order is increased through release of heat energy (increases entropy outside the cell)

Heat is released during . . .

interconversion of different energy types

Potential energy is stored in

chemical bonds, concentration gradients (movement of ions), electric potential (charge)

Gibbs free energy, G

energy of a molecule that could be used to do work

Molecules possess energy because of —-

vibration, rotation, translation, chemical bonds (enthalpy)

Free energy equation

G = H -TS H = enthalpy, T = temp, S = entropy

Change in G determines

if a reaction could occur

Change in G equation

G_products - G_reactants or cH - TcS

energetically favorable

negative change in G, entropy increases, reaction is likely

energetically unfavorable

positive change in G, entropy decreases, reaction unlikely

Many biological reactions decrease entropy, but still occur. How?

Cells couple exergonic (-cG) with endergonic (+cG) reactions so linkage will favor total occurrence (-kcal/mol)

Activated carrier molecules

source of free energy for reactions

When bonds break from input of energy

entropy increases

High energy bonds

releases energy from products stabilizing and increased entropy after the bond breaks

High energy groups and activated carriers

HEG: phosphate, AC: ATP/GTP

HEG: electrons/ H⁺, AC: NADH/FADH₂

HEG: acetyl, AC: acetyl CoA

protein-mediated mechanisms

transfer free energy of phosphoanhydride bonds

Phosphoanhydride bonds

high energy linkage between two phosphate groups, found in ATP

Potential chemical energy

transfer energy to a reactant

Potential mechanical energy

create an energy-rich stressed state

Fixed relationship between

standard free energy and equilibrium constant K

Equilibrium point of reactions

forward and back reactions still occur but concentration is flexible

Steady-state reactions

rate of formation = rate of consumption

Are individual reactions in a cell in equilibrium? Why or why not?

No, cells are not in equilibrium because cells are inherently in chemical disequilibrium, and equilibrium means cell death

50% of non-water molecules in a bacteria are what?

proteins

Why do molecules scale with cell size?

to maintain concentration

guide: 10⁶-10⁹ protein molecules/cell

Copy number

number of identical molecules

• yeast: 50-1000s of copies

• mammal: 10⁵-10⁶ copies

Cells of the same type can vary — —- -

several fold in protein levels

Proteome

complete inventory of all proteins a cell can have

>100,000 proteins made by 22,000 genes

Entire proteome at one time?

No, mammalian cells ~17,000 proteins expressed

Self assembly

extrinsic energy and steric information are not required, intrinsic to molecule in amino acids, primary structure in amino acid sequence specifies 3D structure

hydrophobic effect

drives polypeptide folding, non polar molecules aggregate in aqueous environments to minimize water contact

Secondary and tertiary structure is driven by

hydrogen bonding along carbon chains

Levinthal’s paradox

it would take millions of years for every confirmation to be tested, so only a few confirmations are tested

How are only a few confirmations tested?

• polypeptides seek the lowest point of free energy

• free energy landscape is not flat

• lower free energy = smaller funnel = lower possibilities

function is derived from structure and structure seeks low free energy →

changing structure eliminates function

intrinsically disordered proteins

no higher order state, dynamic ensembles (biomolecular condensates)

Disulfide bonds

covalent cross-links that stabilize extracellular proteins, form in oxidizing environments

Assisted self assembly

not all polypeptides are self-assembled, have proteins but require an assist from a chaperone or chaperonin

Chaperones

binds to polypeptide cotranslationally, assists in folding

Chaperonins

barrel structure that engulfs incorrectly folded proteins, lined with amino acids with hydrophobic r groups, polypeptide turns itself inside out and exits to refold in the cytoplasm

Interacting with a surface or molecule may

prevent inappropriate aggregation, highlight proteins for degradation, and prevent proteins from exploring other confirmations

What are the six types of work that a cell performs?

Synthetic, mechanical, electrical, concentration, heat, bioluminescence/fluorescence

In thermodynamic terms, which one of the following describes the synthesis of a strand of DNA?

The process overall is exergonic, with a negative delta G.

Biochemical pathways are often — - -

far from equilibrium and consist of linked exergonic and endergonic reactions

Resolution

ability to distinguish two close objects

Abbe equation is used

to set microscopes to get high quality images

What is the Abbe equation?

Resolution = 0.61λ / nsinθ λ=wavelength, n=refractive index, nsinθ = numerical aperture

A smaller number as the answer to the Abbe equation means

better resolution

Abbe limit of resolution

minimum possible distance between two objects that can be seen separately, 200 nm/0.2 micrometers with a light microscope

Advantage of electron microscopes

limit of resolution is 0.005 nm, 40,000x better than a conventional light microscope

In practice, EM is about 0.1 (2000x better)

Five steps of preparing a specimen for TEM and the reasons for doing so

1. Fixation (aldehydes) - holds structures in place

2. Dehydration (alcohol) - to keep vacuum clear

3. Embedment (resin)

4. Thin sectioning - plastic embedded cells are sliced

5. Staining (uranyl acetate, O_sO₄) - for contrast, done with heavy metals

Iceman Cometh (CryoEM) method

cells are flash frozen and vitrified

Pros of Iceman Cometh

• no prep, fixatives and stains

• native structure determination

• images are averaged

contrast

relative difference in brightness between object and background

How do we solve the contrast problem in light microscopes?

• differential light absorption (staining)

• enhancement of small changes in refractive index

Stains

absorbs light and reduces amplitude, which dims the light → kills cell

Unstained cells change

position of amplitude peaks by ¼ wavelength

Invented optics for unstained cells

optics shift wavelength another 1/4

Electron microscopy

dead cells

Light microscopy

some are dead, some are live

heat shock proteins

present in normal state as well, chaperone, protect from stress by assisting protein folding, cell compensates in times of stress by adding more

Why does compartmentalization allow for cell growth?

No worries about molecules finding each other

Hydrophobic effect of water molecules

water molecules create a lattice around a hydrophobic molecule - higher order - universe dislikes

Two hydrophobic drops merge and decrease surface area → releases some water molecules from hydrogen bonds → enables greater entropy

phase contrast microscopy

enhances contrast in unstained cells by amplifying variations in refractive index with the specimen, used for living cells, special optics, details are lost

differential interference contrast microscopy

uses optical modifications to exaggerate differences in refractive index, special optics, uses polarized light to show detail