Session 3-4: Intracellular support

Question 1

Why is it important to understand the cytoskeleton?

Answer 1

If we’re able to understand the cytoskeleton, we can use that knowledge to our advantage by enhancing its function in times that we need and inhibiting its function in times that we don’t.

Question 2

Explain the cytoskeleton

Answer 2

It holds up our cells and if we didn’t have a functional cytoskeleton, we wouldn’t be able to perform tasks, such as WBCs clearing infection, wounds wouldn’t close, heart won’t beat, cells won’t divide, cancer wouldn’t metastasize, etc.

Question 3

How does cytoskeleton malfunction impact sickle cell disease?

Answer 3

Sickle cell disease is when there is mutated hemoglobin, BUT the altered oxygen tension leads to an irreversible change in MICROFILAMENT structure which results in permanently sickled cells.

• this means that drugs that stabilize the microfilaments may also be a good method of therapy in addition to gene therapy for hemoglobin mutation.

Question 4

Explain how muscular dystrophy is impacted by malfunction of the cytoskeleton

Answer 4

Muscle dysfunction in muscle dystrophy is caused when the microfilament accessory cross-bridge protein fails to create the necessary lattice structure and leads to disorganization.

Question 5

Alzheimers disease is caused by…

Answer 5

dysfunctional accessory tau protein that lead to microtubule malfunction

Question 6

What causes genetic skin blisters?

Answer 6

Keratin (intermediate filament) mutation leads to skin fragility. (AKA mutation of the cytoskeletal protein)

Question 7

True or false: Only mutations of the cytoskeleton will cause malfunctions in the body/diseases.

Answer 7

False. Dysfunctional accessory proteins will also cause diseases and malfunctions in the body.

Question 8

True or false: the cytoskeleton is not involved intracellular movement.

Answer 8

False.
- vesicles of neurotransmitters move down the axon through a highway of cytoskeleton.
- also moves the cell around its environment.
- transport mechanism for organelles within the cell

Question 9

How does the cytoskeleton impact cellular function/support?

Answer 9

• internal support
• balance between static (support) and dynamic (movement)
• moves organelles within the cell
• moves cell within the environment

Question 10

What are the three fibrous protein networks?

Answer 10

• microtubules (largest)
• intermediate filaments (medium)
• microfilaments (smallest)

Question 11

How are cytoskeletal subunits categorized?

Answer 11

Subunits are based on relative diameter. (NOT LENGTH)
Microtubules (largest)
Intermediate filaments (medium)
Microfilaments (smallest)

Question 12

Explain the way the cytoskeleton can transform based on intracellular needs

Answer 12

Since the cytoskeleton contributes to intracellular support, it can polymerize and depolymerize based on what is needed by the cell.
It can be static for support or it can be dynamic for movement.
- initiates, maintains, and re-organizes cell shape

Question 13

What is the difference between filopodia and lamellipodia?

Answer 13

filopodia - thin projections of the plasma membrane in migrating cells
- caused by bundled polymers w/ cross-linking proteins that drive extension

lamellipodia - sheet-like extensions at the leading edge of cells
- caused by branches polymers that drive extension

Question 14

What are the consequences of actin polymerization in filopodium?

Answer 14

Actin polymerization will cause extension of the membrane into the environment

Question 15

Describe the characteristics of microfilaments: example function, stability, diameter, occurrence, ions associated with polymerization, polar?

Answer 15

• function: membrane support and major projections; cell migration
• stability: dynamic (easily assembled/disassembled)
• diameter: narrowest
• occurrence: in all cells
• monomer type: globular protein (dumbbell shape)
• polymer: F-actin
• polar –> assembles on + end
• polymerization is dependent on monovalent ions, Ca++ and ATP, and accessory “formin” proteins

Question 16

What do you get from actin polymerization?

Answer 16

cell motility
- intracellular polymerization causes extracellular migration
- could be cell extension over wound for healing or invasion of underlying tissue as cancer.

Question 17

How can microfilament structure and function be controlled?

Answer 17

1. polymerization
2. accessory proteins: different kinds of proteins are going to be expressed at different levels in different cells depending on what function needs to be done
   - examples of proteins: myosin motor proteins (cytomotility), kinases (phosphorylation changes the protein shape), structural (vinculin, 2 genes for talin, alpha-actinin)

Question 18

Describe the different structure of microfilaments and how it relates to their function.

Answer 18

bundles (individual, parallel arrays) - tight/dense to support protusions of cell membrane (filopodia/lamellipodia)

networks (gels) - crisscrossing microfilament bundles that provide viscosity to cytoplasm of cell

Question 19

Explain the path of communication of microfilaments in the cell

Answer 19

there is 2-way communication in<=>out
actin fibers <=> accessory proteins <=> integrins <=> extracellular proteins
(if there is a disruption on the INSIDE pathway, there will also be a reflecting disruption on the OUTSIDE, vice versa)

Question 20

What happens when there are different amino acid sequences in different versions of kinases, talins, etc?

Answer 20

There will be variation in their activity or interaction affinity with other proteins
- variations in strength of interactions –> variability in stability of the complex

Question 21

How are actin filaments (microfilaments) involved with viruses?

Answer 21

• HIV actually promotes the polymerization and extension of actin; actin will continue to extend the virus away further and further until the membrane wraps around the progeny virus and gets released from the cell.

Question 22

What does cytochalasin do to viruses (HIV) in relation to microfilaments?

Answer 22

Cytochalasin inhibits actin polymerization (elongation) which means it prevents the HIV virus from being sent out into the cell
- blocks new additions of monomers to the actin filaments by acting as a competitive inhibitor.

Question 23

Is cytochalasin an effective anti-viral therapy?

Answer 23

There will be negative side effects bc actin is involved in a lot of everyday cell processes that include polymerizations.
Cytochalasin inhibits polymerization and is going to inhibit polymerization everywhere else in the body.

Question 24

What are the different subclasses of intermediate filaments (4)?

Answer 24

Multiple proteins (different classes) build intermediate filaments which usually have the same structure/tissue preference
- keratins (skin, hair, nails, epithelia)
- vimentin (fibroblasts)
- neurofilaments (neurons)
- nuclear lamins (in all cells with a nucleus)

Question 25

Characteristics of intermediate filaments: function, stability, diameter, occurrence, polarity?

Answer 25

stability: static diameter: middle of the road; 10nm occurrence: in cells that are subject to stress/pressure or long distances (ex. skin and nerves) polarity? no polarity; monomers can add or remove from any end function: found mostly as polymer/filaments (little monomers); this is because polymer is stable in order to provide MECHANICAL STRENGTH

Question 26

When, why, how would IFs need to be disassembled?

Answer 26

When: during mitosis/cytokinesis Why: the cell is pulling itself apart and the chromosomes need to be divided and chromosomes aren't going to be able to separate through the filament network (cytoplasm division and chromosome separation) How: phosphorylation will drive DEpolymerization to regulate IFs

Question 27

How do IFs attach cells? To what?

Answer 27

IFs attaches the cell to the environment stability of IFs = stability of cell connections (long term connections) hemidesmosomes = cell-substrate connections desmosomes = cell-cell connections

Question 28

Explain EBS (epidermolysis bullosa simplex) and its relation to IFs

Answer 28

- ONE wrong amino acid in protein (mutation in keratin IF) - mutant keratin doesn't provide sufficient resistance to physical stress and cells tear apart, blisters form, epidermis is lost from any physical pressure (IFs provide structural support --> mutated --> reduced structural support)

Question 29

What are characteristics of microtubules (MTs): stability, diameter, occurrence, types of monomers, polarity?

Answer 29

stability: dynamic (easily broken apart and put together) diameter: 25nm (widest) occurrence: different amounts in all cells types: a and b tubulin monomers alternating (polymerization dependent on Mg2+ and GTP) polarity: polar, has directionality (+ end for adding, - end for removing) (addition at the + end can extend the membrane forward) Move things within the cell (like a highway)

Question 30

What are some MT accessory proteins?

Answer 30

- motor proteins = kinesins and dyneins - tau proteins - katanin

Question 31

What is the importance of kinesins and dyneins?

Answer 31

Kinesins and dyneins are motor proteins and they provide intracellular motility

Question 32

Explain the role of tau proteins.

Answer 32

Tau has a LOW level of phosphorylation, which means that it is very compatible with polymerization. - stabilizes the MTs and supports their assembly.

Question 33

What occurs to tau proteins when there is a kinase present?

Answer 33

When a kinase is present, there is hyperphosphorylation --> increases MT disassembly. The hyperphosphorylated tau comes off the MT and the stability generated by the low phosphorylated tau is lost.

Question 34

When there is too much tau phosphorylation that disassembles MTs, what happens?

Answer 34

1. (normal) allows for mitosis; chromosome separation. 2. affects cellular structure (no stability) 3. (negative) MTs are the highways for delivery around an axon, phos-tau disrupts transport of neurotransmitters to the synapse and clumps up to block delivery of signals (alzheimer's)

Question 35

What is katanin?

Answer 35

A microtubule severing enzyme

Question 36

Why are low level of tau good? what do they recruit?

Answer 36

LOW phos-tau recruits FEW katanin, a MT severing enzyme to aid in disassembly. Katanin cuts the MT and creates negative ends. - These negative ends are where monomers and be removed from, which means more disassembly can occur. - This helps aid in normal balance of MT (polymerized/depoly)

Question 37

Why are high levels of phos-Tau bad? What disease does it cause?

Answer 37

High phos-tau recruits A LOT of katanin -> LOTS of cuts to the MT -> LOTS of negative ends -> LOTS of disassembly - no more highway + forms lumps of protein in the way (paired helical filaments) of neurotransmitter pathways = alzheimer's disease - loss of MT and phos-tau filaments impair axonal transport of proteins needed at synapse for signaling

Question 38

What does Taxol do?

Answer 38

Taxol inhibits depolymerization; binds and stabilizes the MT polymer. - TAX(P)OL stabilizes the Polymer - used to treat cancers bc it REDUCES cell division (although it also inhibits the division of healthy cells too)

Question 39

MW of Taxol is too big to travel over the BBB (blood brain barrier). What other options are (almost) available?

Answer 39

EpoD has a MW that is lower than the BBB cutoff - increases the MT stability. - Increased MT integrity -> blocks chromosome separation + improved axonal transport and cognitive performance

Question 40

What does colchicine, colcemid do?

Answer 40

Colchicine binds to the monomer and PREVENTS polymerization (contrast to Tax(P)ol) - treat gout (uric acid build up in joints and WBC come in to clear it out, which causes inflammation); by preventing WBCs from flooding in, there is no inflammation. - disadvantage: WBCs are not able to do other functions (fight infections) (polymerization allows for "highway" for intracellular motility)

Question 41

What do Vinblastine and Vincristine do?

Answer 41

They bind to and form aggregates with the monomer. - We get MORE monomer and LESS polymer over time. - stops cell division by binding to the MT of the mitotic spindle, thereby arresting tumor cells in metaphase (stops mitotic spindle formation)