CELL 360 Exam 1

What determines the resolution of light microscopy?

lambda/2 is the minimum distance that two dots can get before they become one

Wide field Microscopy

all light is visible (whether it's focused or not)

- shows what is IN a cell, but can't discriminate between intracellular and membrane proteins

Confocal microscopy

pinhole focuses light

- good for thicker tissue samples

- identify membrane bound proteins with a cross sectional view

Antigen

a molecule that elicits an immune response--recognized by the antibody

*can have multiple epitopes*

Epitope

portion of the molecule (the antigen) physically bound by the antibody

Parts of an antibody

1. Constant region--same for antibodies in the same class/species, portion recognized by receptors (macrophages)

2. Variable region (also called FAB)--recognizes the antibody/responds to the antigen

**light chain and heavy chain

**gives the antibody specificity

Monoclonal antibody

most efficient cells--cancer cells that reproduce on their own

*antibody targets ONE epitope on an antigen, very specific*

Polyclonal antibody

not as specific--attacks antigen more effectively (more widespread)

*binds to MANY epitopes on an antigen*

Protein sorting on Native Gels

1. Size

2. Charge

3. Shape

Protein sorting on SDS-PAGE

Size only

Protein sorting on Beta Mercaptoethanol

Size only

*will separate dimers*

Co-immunoprecipitation

Do two proteins interact?

-bead binds to the constant region on the antibody, recognizes the protein of interest which then binds to it's dimer

*uses antibodies*

Affinity tags + types

binds to specific types of proteins (co-IP is a subset)

*GST--> glutathione

*His--> cations (like nickel)

*Biotin--> binds to protein side chains

MBP--> amylose resin beads

Three types of chromatography

1. Ion-exchange: based on charge

- + or - charged beads in the column

2. Gel filtration/size exclusion: based on size

-larger molecules take less time to travel because they go around the beads

-smaller molecules take MORE time to go through because they travel through holes in the beads *used for dimers* (they are normally the biggest)

3. Affinity chromatography: antibodies are attached to the beads

-isolate protein of interest based on what sticks to the bead/antibody

Forward genetics

*mutate first, ask later*

- choose phenotype, then randomly mutate the genes

- gene map to find the altered genes that changed the phenotype

Reverse genetics

*identify first, mutate later*

- choose the gene, mutate it, then look for the phenotype change

F-actin

polymer, filaments formed by G-actin coming together

G-actin

monomer of F-actin

Cofilin (actin)

drives disassembly, binds to the - end

Tropomyosin (actin)

stabilizes the actin by binding to each side of it

Tropomodulin (actin)

caps the - end, prevents subtraction, makes the filament longer

Formin + Profilin (actin)

the "basketball game" that aids in nucleation

*Profilin-->binds G-actin onto the filament

*Formin-->stabilizes the actin--forms the arms that attach to profilin

Capz (actin)

caps the + end to prevent addition of monomers

Arp 2/3 (actin)

nucleates and causes branching of the - end *used up after it makes the branches*

Thymosin

inhibits nucleation

Alpha tubulin (microtubule)

never hydrolyzes its GTP (still a GTPase, just not an active one)

Beta tubulin (microtubule)

hydrolyzes it's GTP

GTP bound--> microtubule assembly (favors interaction with alpha tubulin)

GDP bound--> assembly WHEN concentration is 100X higher

What end do microtubules grow from?

the + end

- destabilization happens from this end as well

What end does actin grow/shrink from?

grows from the + end, shrinks from the - end

Gamma-tubulin/gamma-TuRC (microtubule)

nucleates assembly and remains associated with the - end

- 7 complexes that overlap, 13 gamma tubulins for complexes to build on

- forms a gamma-turC or gamma tubulin ring

Gamma-TuSC (microtubule)

small complex part of ring structure

Cilia and flagella structure

DOUBLET--13, 10

*9 doublets on the outside the 2 single microtubules on the inside

Centriole structure

TRIPLET--13, 10, 10

*9 triplets around an SAS-6 core

SAS-6 core--> forms 9 dimers to anchor the microtubules

MTOS or centrosomes

makes new microtubules--contains centrioles in the middle

- gamma tubulin occurs in the gamma ring complexes around the centrioles and tubulin grows from those

MAPS (mictrotubule)

stabilizes + ends and accelerates assembly

- helps regrow the GTP cap (suppresses catastrophe)

GTP cap

keeps the + end stable so it doesn't curve or fall apart

- if the cap isn't refreshed the tubulin will shrink

Types of intermediate filaments

Type 1 + 2--> acidic and basic

- Keratins: made of dimers with one basic group and one acidic group--give strength to epithelial tissues and provide the structure of hair and nails

Type 3-->

- Vimentin: most widely expressed--creates junctions with other cells + does cell signaling

Type 4-->

- Nuerofilaments: helps the axon grow + strengthen it

Type 5-->

- Lamins: special class in the nucleus--makes meshwork--important for gene transcription

Type 6-->

- Nestin: also in axons--regulates axon width

Actin Motors

myosins

Microtubule motors

Kinesin + Dynein

Types of Myosin

Myosin 1--> non processive, + end, vesicle transport

Myosin II--> non processive, + ended, muscle contraction dimer

Myosin 5--> processive, + ended, vesicle transport *dimer*

Myosin 6--> processive, - ended, vesicle transport *dimer*

Kinesin

+ ended, processive, vesicle transport *dimer*

Dynein

- ended, processive, used in vesicle transport

Directionality of neuron vs Epithelial cell

Nueron--> - end towards the nucleus, + end towards the outer edges

Epithelial cell--> + end towards the nucleus, - end towards the outer edges

Adherin (cell-cell)

cadherin (transmembrane) bound to actin

- catenins and vinculin

Desmosome (cell-cell)

non classical cadherins (transmembrane) bound to intermediate filaments - plaques

Hemidesmosome (cell-matrix)

intermediate filaments (keratin) to the ECM

-integrin dimers (transmembrane) and plectin

Focal adhesions (cell-matrix)

bind actin to ECM

- alpha/beta integrin dimers (transmembrane) link to ECM

- talin and vinculin link actin to integrin dimers

Tight junctions

make a tight seal, made of claudins and occludins

- cannot increase tissue strength

Gap junctions

channels made of 6 connexins

- allow SMALL molecules (water, salts, nucleotides, electric current) in the cell but NOT proteins

Nucleolus (what is included and discluded)

connected to side of nucleus--center for making ribosomes and the components

*Histones--> excluded

*Myc (transcrip. factor)--> excluded

*Nucleolin (trascrip. factor)--> FOUND in nucleolus

Nuclear envelope

around the outside of the nucleus--composed of two bilayers separated by the perinuclear space

- continuous with the endoplasmic reticulum

Nuclear lamina

forms meshwork UNDER the nuclear envelope--connected via the LINC complex

- may be connected to heterochromatin/alter transcription

LINC Complex

holds the nuclear envelope in place through nesprin and emerin/sun proteins

*Nesprin--> goes through the first membrane (outer)

*Emerin/sun proteins--> meet the nesprin by going up through the bottom membrane (inside)

What does the nucleoplasm(nucleus) contain? (big picture)

1. Histones

2. RNA poly II

3. mRNA

4. Intron splicing machinery

What does the nucleolus contain? (big picture)

1. rRNA genes

2. rRNA and snoRNA

3. RNA poly I

4. Ribosome assembly machinery

XMAP215 (microtubule)

stabilizes + ends and accelerates assembly

- helps to regrow the GTP cap (suppresses catastrophe)