Cell Biology test 1

Mitochondria function

\-produce ATP via cellular respiration

\-aerobic respiration: utilizing oxygen to generate energy

Mitochondria characteristics

\-Reproduce by division

\-contains their own DNA

\-thought to be an ancestor of bacteria

Endoplasmic Reticulum function

\-make proteins and lipids that are exported

\-translation

Golgi Apparatus Function

\-Modifies proteins from the ER

\-directs proteins to cellular destinations

Peroxisomes

Type of vesicle that is membrane bound and breaks down peroxide (bad for cell)

Lysosomes

Small organelle that recycles or excretes unwanted molecules

Vesicles Functions

\-Transports molecules to specific locations in the cell

\-endocytosis

\-exocytosis

Cell culture benefits

\-accessible to modify

\-controlled environment

\-manipulate DNA

\-Add a drug (pharmacology)

\-one/limited cell types

\-easy to visualize with a microscope

Cell types that can be cultured

\-Embryonic and stem cells

\-neurons

\-muscle

\-epithelium

\-Oocytes

\-Zygotes (IVF)

Cell culture limitations

\-not in their regular tissue/environment

\-cell signaling is limited

\-defined media is constant and not physiologically representative

\-can’t assess tissue-tissue interactions

\-can’t assess interactions with other cell types

How to differentiate between male and female zebrafish

Females are full of eggs and have a little belly bump

Zebrafish benefits

\-Embryonic transparency

\-develop outside mother

\-large brood size

\-sequenced genome

\-conserved genes, anatomy, and physiology

\-Vertebrate

\-CRISPR/Transgenisis

\-closed vasculature

Zebrafish Limitations

\-non-mammalian

\-no in utero work

\-no placenta

\-anatomical differences

What cell bio methods identify proteins?

Immunocytochemistry and Western Blot

How are antibodies used in cell bio and proteins?

\-localize protein in an organism and a particular cell compartment

\-follow protein movement

\-determine under what conditions a protein is made/destroyed

Immunocytochemistry process

1\.) a primary antibody binds to a specific epitope on a protein

2\.) secondary antibody binds to primary antibody

3\.) a fluorociene binds to the secondary antibody

Immunocytochemistry strengths

\-organismal, tissue, cellular, an subcellular resolution

\-Visualize an intact organism/tissue/cell

Immunocytochemistry Weaknesses

\-dead starting material

\-can’t observe protein movement

\-no size information of protein

\-not quantitative (brighter doesn’t mean more protein)

Western Blot Process

1\.) grind up cells to extract proteins

2\.) Gel electrophoresis performed

3\.) Visualize different proteins present

4\.) Blotting— transfer proteins to a different substrate

5\.) Specific protein detection with an antibody

6\.) Visualize protein with antibody attached (only band visible)

Strengths of Western Blot

\-Separate protein

\-Quantitative (darker band = more protein

\-Can detect posttranslational modification (Ex: cleaved)

\-resolution at tissue/cell level

Weaknesses of Western Blot

\-no subcellular resolution

\-cannot visualize protein in a cell

\-can’t detect protein movement in a cell

Polyclonal antibodies steps

1\.) inject purified protein into rabbit (Immune challenge)

2\.) Wait for immune response

3\.) Draw blood and isolate specific antibodies for that immune challenge

Monoclonal antibodies steps

1\.) inject purified proteins (immune challenge) into mouse to produce antibody producing B-lymphocytes

2\.) Fuse a myeloma (cancer) cell with B-lymphocyte to produce hybridoma cells that are immortal and produce antibodies

3\.) culture hybridoma cells to create many cells

Monoclonal Antibody characteristics

\-Cells live forever

\-Endless supply if mAb

\-difficult to make

\-recognize one epitope

Polyclonal antibody characteristics

\-Rabbits don’t live forever— limited supply of pAb

\-need to keep injecting protein

\-easier to make

\-recognizes multiple epitopes

Cell bio methods to identify DNA and RNA

\-CRISPR

\-Transgenisis/GMO

\-*In situ* hybridization

CRISPR functions

Add, delete, and modify genes efficiently

CRISPR mechanism

1\.) introduce single guide RNA that is complementary to the gene of interest

2\.) single guide RNA signals for CAS9 to come and cut DNA

3\.) DNA repairs itself causing a deletion

GMO of Humans using CRISPR steps

1\.) take hormones to hyper-ovulate

2\.) remove eggs from ovaries surgically

3\.) put eggs into Petri dish with sperm

4\.) human embryos form and grow in culture

5\.) genetic modification via CRISPR

Promoters

\-non-coding DNA

\-adjacent to coding gene

\-regulates gene expression

\-vital for timing and location of expression of a gene

*In situ* hybridization (general)

\-localizes mRNA in tissue/cell

\-localizes DNA on chromosomes

\-uses Nucleic acid probes (analogous to abs in Immunocytochemistry)

Chromosomal *in situ* hybridization

\-Chromosomes are marked to localize specific genes

\-DNA

\-Different color for each gene

Strengths of chromosomal *in situ*

\-localizes gene chromosome

\-can show multiple copies of a gene

\-can show translocation of a gene

Weaknesses of chromosomal *in situ*

\-When/where is the gene expressed? (in what cells)

\-What is the resulting protein’s function?

\-Where is protein localized in a cell/tissue/organism?

mRNA *in situ* hybridization

purpose is to localize mRNA in a cell using Nucleic acid probes

Strengths of mRNA *in situ* hybridization

\-shows tissue/cell where gene is expressed

\-shows when gene is expressed

\-organism/tissue/cell is intact

Weaknesses of mRNA *in situ* hybridization

\-doesn’t tell us whether the protein made is localized

\-protein function is unknown

\-not quantitative (darker color doesn’t mean more mRNA)

\-sample is dead

Cre-Lox Recombination System

\-Used to remove a gene by mating two organisms, one with a promoter expressing Cre, and the other has the desired gene flanked by LoxP sites

\-offspring will have gene removed

Cre function

Recombines DNA

Lox

The DNA sequence that Cre binds to and is recombined

What is the Cre-Lox recombination system used to make?

Conditional mutants— gene lost in certain tissues

Reflection

Bouncing of light rays off a surface

Ex: looking at brine shrimp on dissecting scope

Absorption of light

Transfer of light energy to a particle or surface

Ex: fluorescent microscopy, excitation, emission

Refraction

Bending of light rays from one medium to another

Ex: light going through an objective lense, cellular matter, tissue, whole organism

Refraction and magnifying glass

Light rays from sun are refracted by the glass, making the energy concentrated and funnels energy into a smaller area

Confocal vs conventional microscopy light source

Confocal: Laser at a specific wavelength

Conventional: white light with filters

What allows visibility in conventional and confocal microscopy?

Fluorescence

What part(s) of specimen is illuminated in conventional vs confocal microscopy?

Conventional: whole specimen is illuminated

Confocal: light is focused onto a pinpoint on the specimen

Out of focus light conventional vs confocal microscopy

Conventional: out of focus light is not eliminated

Confocal: out of focus light is eliminated

What captures the emitted light in confocal microscopy?

Detector— Photomultiplier tube, PMT

What are the LIVE fluorescent labels?

GFP, CFP, BFP, YFP, RFP

Important cell biological methods used in FRET

Transgenisis, CFP, YFP, and fluorescence microscopy

When does FRET occur?

When CFP is VERY close to YFP (“bound” together; 5 nm)

Purpose of FRET

To determine if 2 proteins or protein modules physically interact

What does FRET detect? What happens with detection?

\-Levels of Ca++ in cells

\-Ca++ concentration is high = conformational change in protein to allow for FRET

\-Ca++ concentration is low = conformational change so that CFP and YFP are not interacting and FRET cannot occur

Role of cytoskeleton in cell function

1\.) correct shape

2\.) mechanical strength to interact with environment

3\.) strong internal structure

4\.) migrate through tissues

5\.) cell division

6\.) respond to signals from other cells

What are polarized cells?

Asymmetrical cells due to movement

Where are actin located in a migrating fibroblast?

At the leading edge

Where are microtubules located

Apical vs basal side of cells

\-Apical: facing the lumen of a tube

\-Basal: attached to underlying tissue

Apical side of cell function

Absorb nutrients

Basal side of cell function

Transfer nutrients to bloodstream

Microvilli function

Increase surface area of cell for more absorption

Adherents junctions functions

Seal epithelium to prevent intestinal contents from entering bloodstream

Cell cortex

Area just under the membrane

Types of intermediate filaments

Desmosomes and hemidesmosomes

Desmosomes function

Adhesive filaments that keep a “sheet” of cells

Hemidesmosomes function

Adhesive filaments that keep the cells attached to the basal lamina

Microtubules characteristics

Polarized and run vertically in cell

What end do microtubules add from?

Plus end

Types of Actin Assemblies as Cytoskeletal Modifying Proteins

\-Stress fiber

\-Lamellipdoium (cell cortex)

\-Filopodium

Stress fibers location and formation

\-towards back of cell

\-contractile bundle

Lamellipodium location and formation

\-Front of cell but not in leading edge

\-Gel-like network that can stretch in any direction

Filopodium location and formation

\-located in leading edge

\-tight parallel bundle that stretches outward

Roles of Cytoskeletal Modifying Proteins

\-Sever

\-Stabilize

\-promote polymerization

\-promote secondary structure of CSK

\-respond to cellular environment (cell signaling)

Filopodia characteristics

\-one dimensional

\-long bundles of actin

\-growth cones of neurons

Lamellipodia characteristics

\-in epithelial cells

\-2D

\-sheets

\-mesh of cross-linked actin

\-growth cones of neurons

Keratocyte

Specialized type of fibroblast that allowed us to learn a lot about cell migration

Keratocyte characteristics

\-Skin

\-Wound closure— 1st cell to location of wound

\-rapid movement when cultured

\-LARGE lamellipodia

\-Rear unattached substratum

How do Keratocytes move?

Treadmilling and retraction of trailing edge

What is treadmilling?

Addition of 3 subunits and then taking off 3 repeatedly to move cell forward

What makes keratocytes treadmill?

A wound signal tells the filaments to polymerize which keeps it fresh and ready to close wounds

What does the “retraction of trailing edge” in a Keratocyte entail?

\-Myosin II contracts actin which is signaled to pull it forward

\-prevents lamellipodial formation at trailing edge

What does cofilin localization do?

Aids depolymerization away from the leading edge

Myosin II function

Contract actin mesh work proximal to lamellipodia

Where is Myosin II present?

trailing edge

Membrane ruffles

Lamellipodia that fail to adhere to substratum

Relationship between cell adhesion and cell movement

\-adhesion is required for movement

\-Degree of adhesion inversely proportional to speed

\-in vivo, cells move on basal lamina or extracellular matrix

What is adhesion?

linkage of CSK to the substratum through cell adhesion molecules

Requirements for cell movement

1\.) traction

2\.) cell forms new attachments

3\.) attachments are stationary as cell moves forward

4\.) trailing edge releases adhesion

\-myosin II pulls cell body forward and releases old cell adhesion attachments

Protein in the Rho family of GTPases

Rho, Rac, cdc42

Characteristics of Rho GTPases

\-Intracellular

\-convert GTP to GDP

\-respond to extracellular cues

\-cytoskeletal rearrangements

What is an active GTPase?

GTP is bound to GTPase

What is an inactive GTPase?

GDP is bound to GTPase

(Made by dephosphorylation of GTP after it’s bound)

What does activating Rho GTPase do?

Stress fibers are made

What does activating Rac GTPase do?

Lamellipodia are made

What does activating cdc42 GTPase do?

Filopodia are made

What is the growth cone of a neuron made of?

Actin

What are axons made of?

Microtubules