biol 201

light microscope

-1000 x magnification
-visible or UV light illumination
-glass or quartz lenses
-eye or camera detection
-brightfield, phase contrast, DIC, widefield fluorescence, composite images, confocal fluorescence

brightfield microscopy

-type of light microscopy
-visible light passes through speciman
-stains needed

phase contrast microscopy

-type of light microscopy
-visible light passes through speciman
-darks darker and lights lighter
-halo around cells

differential interference contrast (DIC) microscopy

-type of light microscopy
-visible light passes through speciman
-darks darker, lights lighter
-shadow around cells

widefield fluorescence microscopy

-type of light microscopy
-uses DAPI(UV-blue) , fluorescein (blue-green), or rhodamine (green-red) to mark cells

composite images

The combining of visual elements from separate sources into single images, often to create the illusion that all those elements are parts of the same scene
-red + green \= yellow
-purple + green \= white

confocal fluorescence microscopy

-type of light microscopy
-shows a 2D optical plane within a 3D perspective
-high detail

tomography

several confocal fluorescence images can be assembled to make a 3D model

coloured stains

H and E, synthetic molecules with high affinity for target and absorbs light

hematoxylin

H, a stain that has a high affinity for DNA and RNA

eosin

E, a stain that has a high affinity for proteins

fluorescent stains

synthetic molecules with an affinity for a target and fluoresces light

fluorescent probes

affinity for target, covalent bond, fluoresces light, ex. antibodies, synthetic

electron microscopy

uses electrons instead of light, the shorter wavelength of electrons gives greater resolution
-20000-100000x magnification
-beam of electrons for illumination
-electromagnets as lenses
-camera for projection
-TEM and SEM

scanning electron microscopy (SEM)

-beam of electrons bounces off speciman revealing 3D image
-sputter coating with metal atoms (uniformally)

transmission electron microscopy

-beam of electrons passes through speciman revealing 2D image
-ultrathin sectioning and metal atom staining

negative staining

specimens highlighted against background of metal atoms
-submerged in metal atoms with atoms surrounding it, but not on it
-outlined

shadowing

specimen sprayed with metal atoms at an angle

freeze fracturing and shadowing

specimen frozen in freon, fractured through its membrane and shadowed, membrane proteins visible in 2 perspectives

collagen

structural protein found in the skin and connective tissue in animals

cellulose

polysaccharide consisting of glucose monomers that reinforces plant-cell walls

absorptive enterocytes

intestinal cells (90%)
-import nutrients

goblet cells

intestinal cells (10%)
-export protective mucus into gut (exocytosis)

paneth cells

intestinal cells (

enteroendocrine cells (EEC's)

intestinal cells (

Eagles Minimal Essential Media

-amino acids
-vitamins
-salts
-glucose
-penicillin
-streptomycin
-phenol red
-horse and human serum

HeLa cells

human epithelial cells of a strain maintained in tissue culture since 1951 and used in research, especially in virology.

subcellular fractionation

A procedure to separate cell components; often includes ultracentrifugation
-homogenization (4 methods)
-isolation
-purification (2 methods)

homogenization

-first step of subcellular fractionation
-breaks the plasma membrane to release cell contents
-m1.) high frequency sound
-m2.) mild detergent to make holes in plasma membrane
-m3.) force cells through small hole using high pressure
-m4.) shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel

isolation- differential centrifugation

-second step of subcellular fractionation
-separates cell components according to whether they sediment or not
-up to 4 centrifuges may be required to isolate desired organelles/macromolecules

purification- velocity centrifugation

-one method for the third step of subcellular fractionation
-purifies cell components according to speed
-fast sedimenting component on bottom, tube pierced at base

purification- equilibrium centrifugation

-one method for the third step of subcellular fractionation
-purifies cell components according to buoyant density
-at equilibrium, components have migrated to a region in the gradient that matches their own density
-concentration gradient of sucrose highest at bottom of tube

enzymes

catalyze covalent bond breakage or formation

structural proteins

provide mechanical support to cells and tissues

transport proteins

carry small molecules or ions, change shape each time they move a cargo molecule
tend to be non-gated

motor proteins

generate movement in cells and tissues

storage proteins

store amino acids or ions

signal proteins

carry extracellular signals from cell to cell

receptor proteins

detect signals and transmit them to the cell's response machinery

gene regulatory proteins

bind to DNA to switch genes on or off

globular proteins

compact, soluable proteins

membrane proteins

cylindrical proteins that have a hydrophobic band around the middle

fibrous proteins

long and thin proteins, most are bundled into huge filaments

transcription factor proteins

positive ones recruit RNA pol (increase gene expression)
negative ones block the promoter (decrease gene expression)

regulation of protein activity

some proteins regulated by phosphates
some proteins regulated by nucleotides
some proteins regulated by activator/inhibitor proteins

Immunofluorescence

method of tagging antibodies with a luminating dye to detect antigen-antibody complexes

direct immunofluorescence

fluorophore is conjugated directly to the antibody molecule that recognizes and binds the molecule of interest

indirect immunofluorescence

An immunofluorescent diagnostic technique in which the fluorochrome is not attached to the primary antibody that recognises the target antigen, but to a secondary antibody that binds the primary antibody.

Fluorescent fusion proteins

genes tagged with GFP in a plasmid makes a protein with GFP

tagged fusion proteins

HA tag on a plasmid with a gene, creates a protein with ha tag (indirect or direct antibody attachment)

gene mutations

cells with a nonfunctioning gene can't make that proteins, occurs before trasncription

morpholinos

synthetic mRNA like molecules bind to target mRNAS and block ribosomes, occurs before translation

antibody depletion

antibodies bond to proteins within the cell and stop them from functioning, occurs after translation

positive control group

event should occur

negative control group

event shouldn't occur

technical controls

allow you to quantify the results of all the groups

SDS-PAGE

denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel
-coat protein in SDS
-load gel (vertical)
-run gel

coomassie blue staining

shows all proteins on a gel
-soak gel in it
-tech control lane 1 for molecular weight markers
tells us band size

western blotting

shows us a specific protein on a gel
-remove gel and transfer to a membrane
-add antibody enzymes
-wash
-ass detection reagents

nuclear pores

holes in the nuclear envelope that allow materials to pass in and out of the nucleus
-import: nucleotides, RNA/DNA pols, cdk/cyclin proteins, TF's, histones, splicosomes
export: mRNA, tRNA, ribosomes

how do nucleotides enter nuclear pores

enter via diffusion
-experiment with gold balls, ultrathin sectioning, stain and TEM
-gold balls enter nucleus only if

test of discovery experiment

experiment based upon observing a process

test of necessity experiment

experiment if you remove something does the process still occur

test of sufficiency experiment

experiment if you add something does the process now occur

nuclear localization signal

The signal sequence for the nucleus that enables proteins to move through pores in the nuclear envelope.

requirements for nuclear import

-nuclear localization signal
-importin proteins

importin

transport receptor that binds to the NLS of the cargo and interacts with the nuclear pore
-high affinity for nuclear pore
-high affinity for cargo in cytosol, low in nucleus

requirements for nuclear export

-nuclear export signal
-exportin proteins

exportin

nuclear receptor protein that binds to the nuclear export signal of proteins in the nucleus and then transports the bound protein out through the nuclear pore complex and into the cytosol
-high affinity for nuclear pore
-high affinity for cargo in nucleus, low in cytosol

ran-gtp

binds importin causing it to dissociate from the cargo
binds exportin causing it to bind its cargo

ran-gdp

formed after Ran-GTP hydrolyzes itself in the cytosol, then dissociated from the receptor

membrane lipid synthesis and delivery

enzymes in ER synthesize phospholipids, glycolipids and cholesterol
-delivery to ER and nuclear envelope via lateral diffusion
-delivery to the rest of endomembrane system via transport vessicles

phospholipid exchange proteins

used to move synthesized membrane components from the ER to other organelles in the cytoplasm
-mitochondria, chloroplasts and peroxisomes

protein synthesis and delivery

some made by free ribosomes then imported, some made at ER
-membrane ones have ER signal and transmembrane domain(s), ribosomes at ER then lateral diffusion

vesicles

move material within endomembrane system, exocytosis and endocytosis
-formed via cargo receptors, coat proteins, adaptin and dynamin

exocytosis

export of cell products
-secretory vesicle to extracellular space

endocytosis

import of material
-extracellular space to vesicle or vacuole

phagocytois

importing whole cells

receptor mediated endocytosis

importing whole proteins

cargo receptors

cargo ligands will stick to these in vesicles

clathrin

a coat protein for the trans golgi network, lysosme and extracellular space in vesicles

coat protein

a protein that surrounds a membrane vesicle and facilitates vesicle formation
-assemble into cages

cop 2

a coat protein for vesicles destined to the ER

cop 1

a coat protein for vesicles destined for the golgi

adaptin

connects cargo receptor to coat in vesicles

dynamin

closes the vesicle

transferrin

protein synthesized in liver cells
-carry iron in blood
-carry iron into future RBC's using receptor mediated endocytosis

vesicle movement

use microtubules to move

kinesin

a microtubule motor protein that walks inside to outside (- to +)

dynein

a microtubule motor protein that walks outside to inside (+ to -)

SNARE's

membrane proteins that mediate vesicle fusion

v-SNARE

specialized protein anchored to vesicles to aid their fusing to target

t-SNARE

specialized protein anchored to the target membrane to bind v-SNAREs to dock vesicles, making them ready for release

golgi cis face

receiving side of golgi apparatus

golgi trans face

shipping side of golgi apparatus

animal cell golgi apparatus

one large sack

plant cell golgi apparatus

many little sacks

golgi apparatus

modify and distrbute proteins and membrane lipids made in the ER

constitutive exocytosis

constant stream of transport vesicles off the trans Golgi that delivers newly made lipids and proteins to the plasma membrane

regulated exocytosis

• operates in cells specialized for secretion
• membrane fusion occurs only in response to an extracellular
signal