BFCP S03 + S04

S03: Intro Cell + Tools of Cell Bio; S04: Cytoskeleton

T/F: all cells contain the same copies of the genome with minor exceptions

T

If all cells have the same genome, how do they perform different functions?

They differentiate based on how they use genetic instructions/cell signaling

What is the least complex eukaryote that we commonly use as a model organism?

Yeast

Why are yeast a good model organism?

1. Homologous proteins: similar in yeast and humans

2. Easy to grow and manipulate

Nematode C. elegans significance

First multicellular organism to have its complete genome sequence determined

Zebrafish are ideal for ____ studies because ______

developmental studies because their embryos develop outside of the mother and are transparent

Significance of cell culture

Is it usually one or multiple cells being harvested?

Allows for a controlled environment in vitro

Usually single type of cell

Flow cytometry purpose

• Provides quantitative information on individual cells

  • Whether the target is present and at what quantity

• Label cells with fluorochrome for a target molecule and allow them to flow through

• All cells will scatter light

• The cells with the target molecule scatter light AND emit fluorescence

Flow cytometry

Fluorescence activated cell sorter (FACS) purpose

To separate cells with a specific characteristic from a mixture using “gating”

• Cells with a specific target will have fluorescence

• Each cell droplet is given a negative charge proportional to the amount of target of interest present

  • More target of interest = more fluorescence = more negative electrical charge

• Magnetic plates will sort cells

• Cells can be “gated” and software can sort out the gated cells

Fluorescence activated cell sorter (FACS)

Cell lysis purpose

To study cells at the subcellular and molecular level

• Rupture cells

• Centrifuge the cell components by size and density

  • Heaviest precipitates first

Cell lysis

Antibodies are made up of WBCs called ______ and are (very specific/not specific) to their antigen.

B-cells

very specific

How can we produce antibodies of interest in animals?

1. Inject foreign protein of interest into animal

2. Animal creates antibodies against protein of interest

Method of extracting polyclonal antibodies

Harvest animal blood serum with a mixed combination of all antibodies produced

To make monoclonal antibodies, take ______ from the animal. Fuse these with a _____ to create a hybridoma.

To make monoclonal antibodies, take B-cells from the animal. Fuse these with a tumor cell lineH to create a hybridoma.

Hybridoma

A fused B cell and tumor cell that continuously produces monoclonal antibodies

T/F: light microscopy can be done on live cells

T

Brightfield light microscopy

Light is transmitted straight through the specimen

Phase-contrast light microscope

Makes highly transparent objects more visible

Differential-interference-contrast light microscope

Exaggerates differences in density

Using light and 2 sets of filters to excite fluorochromes and visualize their wavelength

Fluorescence microscopy

Explain the following methods of tagging molecules of interest in fluorescence microscopy. Indicate if they can be done live:

1. Genetic tagging

2. Epitope tagging

3. Immunolabeling with antibodies

1. Genetic: adding GFP to proteins of interest and tracking LIVE

2. Epitope: adding an epitope tag to a protein and detecting it via antibody binding to epitope (usually fixed)

3. Immunolabeling: fluorescence secondary antibody binds to primary antibody which binds to target (only fixed)

Laser scanning confocal microscope

Fixed or live?

Using lasers to focus on a very specific point and combining data to create a 3D structure. Usually on fixed cells.

T/F: electron microscopy can be done on live cells

F - only fixed samples

Transmission electron microscopy

Beams of electrons go THROUGH the target to see ultrastructural details

Scanning electron microscopy

Beam of electrons onto the sample surface

What is the benefit of electron microscopy > light microscopy?

Can reveal much smaller structures and in greater detail

What are 3 methods of following protein dynamics?

1. Fluorescence resonance energy transfer (FRET)

2. Fluorescence recovery after photobleaching (FRAP)

3. Photoactivation

• Tag 2 proteins with different fluorescents

• Excite protein X with the appropriate laser and observe if protein Y emits light

• Purpose: to determine if 2 proteins interact

Fluorescence resonance energy transfer (FRET)

• Tag protein of interest

• Use a laser to bleach the fluorescence

• Watch how other labeled proteins diffuse throughout this area by tracking the reappearance of fluorescence

• Purpose: to understand the protein’s kinetic parameters

Fluorescence recovery after photobleaching (FRAP)

• Photoactivate protein of interest with a wavelength of light

• Watch how that fluoresced target protein move around/away from that spot into other regions

• Purpose: to understand the target protein’s kinetic parameters

Photoactivation

3 parts of the cytoskeleton from smallest → largest, and identify if they are polar or nonpoar

1. Microfilaments (actin filaments): polar

2. Intermediate filaments: nonpolar

3. Microtubules: polar

Which part of the cytoskeleton is the toughest and most durable, having the greatest tensile strength against mechanical stress?

Intermediate filaments

Where are intermediate filaments found?

Throughout the cytoplasm - around/inside the nucleus and extending to the cell periphery

Which cytoskeleton part is found underlying and strengthening the nuclear envelope?

Intermediate filaments

Which cytoskeleton part is responsible for limiting the extension of cell stretching and pulling?

Intermediate filaments

Intermediate filaments have a ______-like shape

rope-like

The structure and composition of intermediate filaments

Long fibrous proteins that form tetramers. 8 tetramers form a long rope

The following are examples of which cytoskeleton part: keratins, desmins, vimentin, neurofilaments, nestin, nuclear lamins

Intermediate filaments

Keratin is an example of ________ and is found in ____ cells.

Keratin is an example of an intermediate filament and is found in epithelial cells.

Lines and strengthens the inside surface of the nuclear envelope

Nuclear lamina

The nuclear lamina is made up of ___________.

intermediate filaments

How are IFs stabilized and reinforced into bundles?

With accessory proteins like plectin

Epidermolysis bullosa simplex (EBS)

Mutations in keratin genes that encode IFs cause the skin to be highly vulnerable to mechanical injury

Which cytoskeleton part acts as an intracellular “highway” to move cell components?

Microtubules

Explain the dynamic instability of microtubules

They can rapidly disassemble in one location and reassemble in another

Microtubules grow out from ______

centrosomes

Microtubules are composed of dimers of ___ (+/-) and ____ (+/-) tubulins. Each microtubule has __ protofilaments.

Microtubules are composed of dimers of a (-) and b (+) tubulins. Each microtubule has 13 protofilaments.

The centrosome is composed of 2 ____ surrounded by pericentriolar material. It is located (close/far away) from the nucleus, except when the cell is in mitosis.

2 centrioles

close

Microtubules grow from the __________ ring complex on centrosomes that serve as the nucleation site for the growth of microtubules.

y-tubulin ring complex

Centrosome function

Controls the production and location of microtubules

Explain how microtubules are built using GTP

Tubulin dimers that are bound to GTP are added to the + end of a growing microtubule. Shortly after addition, they are hydrolyzed into GDP.

GTP cap

The end of a microtubule, where the dimers are still attached to provide stability to the molecule

What would happen if there was no GTP cap on a microtubule?

Rapid depolymerization due to the instability of GDP

Microtubule-associated proteins (MAP)

Regulates the organization of microtubules

Prevents depolymerization when the GTP cap is lost by providing stability

Motor proteins use energy from _______ to travel along _____

ATP hydrolysis

microtubules

Transport of 2 microtubule motor proteins: kinesin and dynein

Kinesin: moves towards the + end, away from the middle/centrosome

Dynein: move towards the - end, towards the middle/centrosome

Kinesins and dyneins have 2 ___ on one end that attach to ____ and hydrolyze ____.

Kinesins and dyneins have 2 globular heads on one end that attach to microtubules and hydrolyze ATP.

Kinesins and dyneins have 1 ___ that interacts with _____.

Kinesins and dyneins have 1 tail that interacts with cargo.

The propulsion of a globular head of a motor protein forward is driven by _____

The binding of ATP on the other head

Kinesins tend to move the organelle _____ outward, while dyneins tend to move the organelle ____ inward.

Kinesin moves the ER

Dynein moves the Golgi

Effects of colchicine treatment

Causes microtubules to disassemble, changing the locations of the ER and golgi

Cilia and flagella are composed of ______ and use ____ to drive their movement.

Cilia and flagella are composed of microtubules and use dynein to drive their movement.

The definition and structure of the axoneme

9+2 structure: 9 outer doublets of microtubules, and 2 single microtubules in the middle

Found in cilia and flagella

The microtubule doublets in cilia/flagella ___ to allow for movement, while linker proteins ______

bend

linker proteins keep the doublets paired while sliding

T/F: dynein “walks” along cilia/flagella to move them

F: we don’t want the microtubule doublets to separate, so the microtubules bend instead

What powers the locomotion of cilia and flagella?

ATP hydrolysis

What do microtubule-specific drugs target?

Prevention of microtubule polymerization/depolymerization

Which cytoskeleton part is responsible for cell movement and the flexibility of cell shape?

Microfilaments

Actin filaments are also called ______

microfilaments

Where are microfilaments located?

Around the cell, but mostly in the cell cortex

Structure of microfilaments

Actin monomers polymerize to form actin filmanets

Microtubule polymerization involved the hydrolization of _____, while microfilament polymerization involves the hydrolization of _____.

Microtubule polymerization involved the hydrolization of GTP, while microfilament polymerization involves the hydrolization of ATP.

What is actin treadmilling?

When ATP-bound actin is added to an actin filament on the + end at the same rate as ADP-bound actin is being removed from the - end.

Actin-binding proteins function

What controls these proteins?

Where and when actin filaments form and grow

Controlled by extracellular signals

Actin combining with ____ allows it to form the sarcomere structure and contract muscles.

myosin

Actin filaments concentrated in the cell cortex allow for _______.

support of the outer surface and mechanical strength

Explain how cell crawling depends on actin using lamellipodia and filopodia, and myosin

-Actin polymerization on the protruding end allows the cell to push forward

-Lamellipodia forms protrusions on the plasma membrane to move the cell forward

-Filopodia detects cues to guide the cell

-Contraction ofm yosin on the rear end of the cell draws the cell forward

Microvilli are made up of ________.

actin filaments