Bis104 M1

Carcinoma

Cancer epithelial cells

Sarcomas

Cancer of connective tissue

Leukemia

Cancer of the blood

Lymphomas

Cancer of immune cells

Benign

Tumor constrained in place

Malignant

Tumor invades tissue which leads to metastasis

Metastasis

When the cancer enters the bloodstream and moves to a distant organ

Cancer tumor traits

Invasive and lost control of division, all cells from a tumor are derived from a single cell

Carcinogens

Chemical that can lead to cancer

Tumor suppressor genes

Genes that prevent cell proliferation (brakes), need to lose both copies for loss of function

Protooncogenes

Normal genes that tell cells to divide, under tight control (Accelerators), only need one mutated copy to increase activity

Oncogenes

The mutated form of protooncogenes

Ways of studying cells

Microscopy, genetics, biochemistry

Necessary (when studying genes)

When the gene is removed, there is a change. Therefore, the gene is ___

Sufficient (when studying genes)

When the gene is added, there is a change. Therefore, the gene is _____

Antibodies

A specific reagent that can bind to a specific protein

Immunoglobin G (IGGs)

A commonly used antibody, contains two light chains and two heavy chains that are connected by disulfide bonds

FaB region

The region on IGGs that have variable domains and binds the antigen. Very specific, the “ends” of the Y-shape

Fc region

The region on IGGs that has a constant domain. The “base” of the Y-shape

Western blots

Used to determine if a protein is present or not, how much, and its MW

Steps to Western Blot

1. Lyse the cell by boiling in SDS —> solubilizes membranes, unfolds proteins, and coats it in a negative charge

2. Separate the protein by size on SDS PAGE, visualize proteins with cuomasie blue stain

3. Transfer the proteins from gel to a membrane

4. Probe the membrane with an antibody to see just the protein of interest

Immunoprecipitation

A way to isolate a single protein, needs the antibody against the specific protein

Immunoprecipitation steps

1. Lyse cells with Donce Homogenizer (physically rips open cell but keeps organelles and protein complexes together)

2. Clear lysate with centrifugation, separating insoluble (pellets) and solubles (supernatant)

3. Incubate supernatant with antibody

4. Purify antibody/antigen complex by centrifuging out bead + protein + IGG + antigen and washing pellet

5. SDS PAGE and cuomasie stain to visualize protein

HER2 receptor

A receptor that normally tells cells to divide, but extra copies lead to breast cancer from uncontrolled division

Monoclonal antibody

Antibody engineered in a lab that targets a specific antigen, used when treating HER2

Subcellular fractionation

A method for studying cells that separates organelles

Subcellular fractionation steps

1. Lysis of cells with a dound homogenizer so organelles stay intact

2. First spin at low speed (600xg)

3. Spin supernatant at medium speed (15,000xg)

4. Spin supernatant at high speed (100,000xg)

5. Centrifuge through sucrose density gradient at 65,000xg to view bands

Organelle(s) present in pellet after low speed (600xg)

Nucleus

Organelle(s) present in supernatant after low speed (600xg)

Everything besides nucleus

Organelle(s) present in pellet after medium speed (15,000xg)

Mitochondria, lysosomes

Organelle(s) present in pellet after high speed (100,000xg)

Golgi, plasma membrane, ER

Organelle(s) present in supernatant after high speed (100,000xg)

Cytosol

Limit of resolution (d)

How far apart two objects should be to resolve them

Fluorescence microscopy

Microscopy that uses fluorescent molecules as markers

Indirect immunofluorescence

Using a secondary antibody that recognizes the primary one attached to the protein of interest and fusing it with a fluorophone to study it

Green fluorescent protein (GFP)

Protein found in jellyfish that emits a green light when exposed to UV light, allows us to track a protein/gene of interest

Electron microscopy

Higher resolution microscopy, use magnets to focus on electrons at accelerated high voltage , can see the lipid bilayer, requires thin and dead samples and lots of stains

Core concepts when studying cell biology

1. Localization of protein (where it is) tells you its function

2. Proteins work in complexes + networks

3. Cell processes are dynamic

Phosphoglycerides

Most common lipid in membranes, has phosphate head, glycerol backbone, and fatty acid tail

Spingolipids

Slightly longer lipid that phosphoglycerides which lead to a thicker membrane

Rafts (in membranes)

Patches of sphingolipids

Sterols

Component of membranes that stiffens them and makes them less permeable at the head, OR spaces out the tails to make it more fluid

Important properties of membranes

1. Package in energetically favorable way

2. Self-assemble/reseal after breaking

3. Very fluid (flex, rotate, and flip flop)

4. Contain proteins

Flippase

Enzyme that flips phospholipids (energetically unfavorable)

FRAP

Studying membrane proteins by adding GFP to a membrane protein, photobleaching with light, and observing fluorescent recovery (spot gets greener)

Single-pass alpha helix

Membrane protein that goes through membrane domains on each side, 20-22 hydrophobic amino acids, around 6-7 turns in membrane

Hydrophobicity plot

Measures how hydrophobic/philic protein sequence is from N to C terminus, spike in hydrophobicity indicates the area that passes through the membrane

Beta-barrels

Membrane protein made from beta-sheets, makes a hole in membrane

Lipid modified proteins

Membrane protein that gets lipid added post-translationally that drives it to the membrane

Peripheral proteins

Proteins outside of the membrane, attached to membrane proteins by noncovalent bonds, can be removed with high salts

Non-ionic detergents

A way to mildly solubilize membranes so they retain function

Ionic detergents

A way to denature and unfold proteins, breaks down the membrane so it can be analyzed

Trypsin

Enzyme that digests other proteins, can’t cross the plasma membrane, used when analyzing proteins and their locations

[K+]

More inside the cell

[Na+]

More outside the cell, balance K+

[Cl-]

More outside the cell, balance Na+ and negative charges inside the cell

[Ca++]

More outside the cell, used for signaling

Passive transport

Using diffusion to move high concentration to low concentration (down a gradient)

Gated-channels

Assist in passive transport, specific for an ion, opened by a signal, bi-directional

Facilitated transporters

Assist in passive transport, specific, let larger molecules through, conformational changes

Active transport

Couples energy with conformational changes in a protein to pump against gradient

Symporter

Co-transporter where ions go in the same direction (one against and one down the gradient)

Antiporter

Co-transporter where ions go in opposite directions

Protein translocation

When a protein being made simultaneously crosses the ER membrane, used when protein is going to be secreted

Signal sequence

The “address” for a protein that targets them to the ER/secretory pathway, ~8 hydrophobic amino acids

Cell free systems

A method for studying cells/proteins, where everything needed for translation is in a test tube

Signal recognition particle (SRP)

Entirely cytoplasmic, a complex of proteins and 1 RNA, contains three ligands: ER signal peptide, SRP receptor, and GTP nucleotide

SRP receptor

An integral membrane protein in the ER membrane that binds to SRP + GTP/ATP, tells the SRP “we’re at the ER”

Translocon

Gated protein conducting channel in the ER membrane, protein made through channel into the ER during translocation (ex. Sec61)

Signal peptidase

Enzyme that cleaves off the signal sequence once the protein is made in the ER

G proteins

Enzymes that use energy from GTP hydrolysis to ensure reactions occur in the correct order, includes switches and timers

GTP Exchange Factor (GEF)

Induces GDP out and GTP in on g-protein, switch that causes conformational change between GDP and GTP states

GTPase activating protein (GAP)

Acts as a timer by accelerating the activity of g-protein

Chaperones

Assist in protein folding

Type I Transmembrane protein

Single-pass, N-terminus in the ER, C-term in the cytoplasm, has stop transfer signal

Stop transfer signal

~20 hydrophobic amino acids in transmembrane proteins that signals for lateral transfer by translocon so the rest of the protein can be made in the cytoplasm

Type II Transmembrane protein

Single pass, N-term in cytoplasm, C-term in ER, has start transfer sequence

Start transfer sequence

~20 hydrophobic amino acids in transmembrane proteins that signals for SRP to bind and translate the rest in the ER

Type III Transmembrane protein

Single pass, N-term in ER, C-term in cytoplasm, internal signal sequence so N-term still ends up in ER

Internal signal sequence

~20 hydrophobic amino acids in transmembrane proteins that signals for SRP to bind and take N-terminus to ER post-translationally (already made in cytoplasm)

Type IV Transmembrane protein

Multi-pass, most common transmembrane protein, can have signal sequence or not, look for lots of + amino acids on cytosolic side to identify

N-linked glycosylation

The covalent attachment of sugars onto specific asparagine residues, stabilizes extracellular proteins

Steps for glycosylation in ER

1. Make sugar-free precursor in cytosol(2 NAG and 5 Mannose)

2. 2 NAG-5 mannose flipped into ER by flippase

3. Add more sugars (2 NAG, 9 Mannose, 3 glucose total)

4. Complex transferred to Asp by oligosacchryl transferase

5. 2 glucose trimmed before exiting ER

6. Once fully folded, the last glucose is trimmed off

Basic steps for vesicle transport

1. Budding

2. Docking

3. Fusion

Coat proteins

Proteins that form a cage around the cytosolic face of a vesicle, discarded before fusion to membrane

COPII Coated vesicles

Vesicles that move from ER to Cis-golgi, anterograde

COPI Coated Vesicles

Vesicles that move from the golgi to the ER or within the golgi trans to cis, retrograde

Clathrin coated vesicles

Vesicles that move from the trans-golgi to endosomes (anterograde), and plasma membrane to endosomes (retrograde)

Steps to coating/vesicle formation

1. Recruit a g-protein (Sar1 for COPII) to the ER membrane, changed to Sar1-GTP

2. Sar1-GTP recruits 4 proteins (= COP II Complex), forms a cage and curves the membrane —> budding

3. A GAP (ex. Sec23) causes change to Sar1-GDP after ~30 seconds, Sar1 and COPII complex falls off

4. Uncoated vesicle can now go to destination (ex. golgi for COPII) with V-SNARE exposed

SNARES

Complexes of 4 long alpha-helices that wind together like a rubber band, allow specificity for docking and mediate fusion

V-SNARES

SNARES that are integral membrane proteins in the vesicle (2 per)

T-SNARES

SNARES that are integral membrane proteins in the target membrane (2 per)

Rabs

G-protein that mediates fusion, allows tethering to target membrane in GTP form for a brief time

KDEL

An ER retention signal in BiP that allows it to return back to the ER after escape

Golgi

Stacks of cisternae, contains cis, medial, and trans golgi with different resident enzymes, vesicles are modified as they move through the different layers

Trans-golgi network (TGN)

The “far” side of the golgi where vesicles exit, acts as a sorter and targets vesicles to different places

Vesicle transport

A theory for how proteins move through the golgi, theorizes that cargo stays in vesicles that move cis to trans and the cisternae are stable

Cisternal maturation

A theory for how proteins move through the golgi, theorizes that vesicles and enzymes move backwards while cargo stays in moving cisternae, new cisternae are made and mature, more popular theory in the field

Lysosomes

Organelle responsible for breaking down/processes large objects/organelles (interchangeable with vacuoles)

Phagocytosis

Bringing large objects into the cell and chopping them up