Cytoskeleton

Question 1

Cytoskeleton

Answer 1

A dense network of protein filaments that crisscrosses the cytosol and provides support to cell membranes. It helps the cell to maintain its shape and move around

Question 2

What is the cytoskeleton made of?

Answer 2

Protein filaments. Each type of protein filament is assembled from certain subunits reversibly so that cells can assemble or disassemble the filaments if they need to. The filaments are polymers whose assembly is regulated.

Question 3

How can the cytoskeleton be visualized? (2 ways)

Answer 3

1. Electron microscopy. Detergents are used to make the plasma membrane and organelles soluble and to release the cytoplasm
2. Immunofluorescence microscopy. Provides colored networks of each type of protein

Question 4

3 protein filament systems of the cytoskeleton

Answer 4

1. Microfilaments
2. Microtubules
3. Intermediate filaments

Question 5

Microfilaments

Answer 5

Polymers of actin that are organized into networks by actin binding proteins. Form rigid gels and linear bundles as well. They organize the plasma membrane and give support to surface structures like microvilli. They also serve as tracks for motor proteins and work with myosin to provide contractile function, like a muscle

Question 6

Microtubules

Answer 6

Rigid tubes formed from the protein tubulin, organized by microtubule associated proteins. They make up the mitotic spindle that is necessary for cell division. Organize organelles and support cilia. Motor proteins (kinesins and dyneins) transport cargo along microtubules for long range transport of organelles.

Question 7

Motor proteins

Answer 7

Proteins that use ATP hydrolysis to generate linear or rotational motion. They use microfilaments as tracks and use microtubules to transport cargo.

Question 8

Intermediate filaments

Answer 8

Polymers made from tissue-specific subunits and assembled into pre-existing filaments. Located on the inside of the nucleus. They give support to the cell membrane and give structural integrity to cells in tissues. They have structural and barrier forming functions in hair, skin, and nails. They are not used as tracks by motor proteins and are less dynamic that microtubules and microfilaments.

Question 9

Actin filaments and epithelial cells

Answer 9

Actin filaments are found on epithelial cells and can be visualized on microvilli. The apical and basal domains of epithelial cells are responsible for transporting nutrients. Nutrients are moved from the lumen at the apical surface through the basal surface to the bloodstream. Therefore, the apical and basal domains of epithelial cells require multiple transport proteins to function correctly.

Question 10

How do cells use actin filaments? (2)

Answer 10

1. Dynamic bursts of actin filament assembly power the movement of endocytic vesicles away from the plasma membrane
2. During mitosis when organelles have been duplicated and segregated, actin forms a contractile ring to constrict and generate 2 daughter cells

Question 11

How are microfilaments arranged in the cell?

Answer 11

There can be multiple arrangements of microfilaments within a single cell.

Question 12

Actin

Answer 12

An abundant intracellular protein in eukaryotic cells. Their abundance depends on their location in the cell or tissue. Actins have been classified into alpha, beta, and gamma categories based on their charge.

Question 13

G-actin

Answer 13

A globular monomer of actin. G-actin is separated into 2 lobes by a cleft. There is a site for ATP and magnesium to bind at the base of the cleft, called the ATPase fold. When ATP or ADP bind to G-actin, the nucleotide changes the conformation of the molecule. G-actin can polymerize to form F-actin in a reversible reaction.

Question 14

F-actin

Answer 14

A filamentous polymer of actin, which is a linear chain of actin subunits. F-actin is a major component of microfilaments.

Question 15

Polarity of actin filaments

Answer 15

All subunits in an actin filament are oriented the same way, so the filament exhibits polarity (one end is different from the other). The positive end is where actin subunits are added, which is also where the ATP binding cleft of the terminal actin subunit comes in contact with the neighboring subunit. The negative end is where subunits are removed.

Question 16

Which electron micrography finding demonstrates the polarity of actin subunits?

Answer 16

The myosin S1 head domains bind to actin subunits in a specific manner. When bound to all the subunits in a filament, S1 looks like it’s spiraling around the filament, forming arrowhead-like decorations. The negative end is pointed and the positive end is barbed

Question 17

3 phases of G-actin polymerization in vitro

Answer 17

1. Nucleation phase
2. Elongation phase
3. Steady-state phase

Question 18

Nucleation phase of actin polymerization

Answer 18

There is a lag period in which G-actin subunits combine into an oligomer of 2-3 subunits. Once the oligomer gets to 3 subunits, it can act as a seed for the next phase.

Question 19

Elongation phase of actin polymerization

Answer 19

The short oligomer rapidly increases in length as actin monomers are added to each of its ends. As the F-actin filaments grow, the concentration of G-actin filaments decreases until there is equilibrium between the filaments and monomers. The system reaches a steady state

Question 20

Steady-state phase of actin polymerization

Answer 20

G-actin monomers exchange with subunits at the filament ends, but there’s no net change in the total length of the filaments

Question 21

Treadmilling

Answer 21

The two ends of a myosin decorated actin filament grow at different rates. Treadmilling occurs when ATP-actin subunits are added faster at the positive end than the negative end of the actin filament. This causes a lower critical concentration at the positive end and treadmilling at a steady state. Once the molecule gets to a steady state, subunits are now added to the positive end and the negative end has a low concentration- this is treadmilling. Only occurs in a test tube, not in situ cells. Can occur in microtubules as well.

Question 22

Cytochalasin D

Answer 22

A fugal product that poisons actin by depolymerizing actin filaments. It binds to the positive end of F-actin and inhibits addition of subunits. The actin cytoskeleton will disassemble and cell movements like locomotion and cytokinesis are inhibited.

Question 23

Phalloidin

Answer 23

Used to localize F-actin in fluorescence microscopy. It is a mushroom toxin that binds at the interface between subunits in F-actin, which locks the subunits together and prevents actin filaments from depolymerizing. Many processes depend on actin filament turnover, so phalloidin causes these systems to no longer work and the cell dies.

Question 24

Rate limiting step of actin polymerization

Answer 24

The formation of an initial actin nucleus from which a filament can grow. This step is a control point in cells, which determines where actin filaments are assembled and what type of actin structures can be generated

Question 25

Formin

Answer 25

A protein that regulates actin assembly through signal transduction pathways. Formin leads to the assembly of long actin filaments. The FH2 formin domain forms a donut shaped complex to bind two actin subunits. It holds the subunits so that the positive end of the newly synthesized filament is pointing toward the FH2 domain. The filament will grow at this end with the FH2 domain still attached.

Question 26

Profilin

Answer 26

Profilin can exchange the ADP nucleotide on G actin for ATP to generate profilin-ATP-actin. The FH1 domain of formin acts as a landing site for profilin-ATP-actin to increase the concentration of these complexes. The actin from the profilin-actin complexes is fed into the FH2 domain to add actin to the positive end of the filament, and profilin is released. Studied using optical tweezers

Question 27

Thymosin

Answer 27

Cells typically have a very large amount of G-actin, but the actin does not polymerize. This is because of thymosin. It binds to ATP-G-actin, which inhibits addition of the actin subunit to either side of the filament. It acts as a buffer of unpolymerized actin, making ATP-actin subunits available as needed.

Question 28

Capping proteins

Answer 28

Cells can regulate treadmilling and actin filament dynamics using capping proteins that bind to filament ends. There are 2 classes- one that binds positive ends and one that binds negative ends. Without capping proteins, the filaments would continue to grow and disassemble in an uncontrolled manner

Question 29

CapZ

Answer 29

A capping protein that binds with high affinity to the positive end of the actin filament. It inhibits subunit addition or loss. Its activity can be inhibited with regulatory phospholipid PI(4,5)P2. Also, some regulatory proteins are able to bind the positive end and protect it from CapZ, while still allowing assembly there if necessary.

Question 30

Tropomodulin

Answer 30

A regulatory protein that inhibits filament assembly and disassembly by binding to the negative end of an actin filament. Found in cells (like RBCs) where actin filaments have to be stabilized for long periods of time. It works with tropomyosin to stabilize the filaments

Question 31

Severing proteins

Answer 31

Proteins that sever actin filaments. One protein, gelsolin, is regulated by calcium concentration. Once it binds calcium, gelsolin undergoes a conformational change and inserts itself between the subunits of the actin helix, breaking the filament. It caps the positive end, creating a new negative end that can disassemble

Question 32

Actin nucleation

Answer 32

This is accomplished by the formin FH2 domain. Its forms a dimer and nucleates filament assembly. The dimer binds two actin subunits, and by rocking back and forth, it can allow additional subunits to be inserted between the FH2 domain and the positive end of the growing filament. FH2 protects the filament's positive end from being capped by capping proteins

Question 33

Optical traps

Answer 33

Used to study molecular motor proteins such as myosin.

Question 34

Listeria

Answer 34

Hijacks normal cell motility processes- if actin filaments become immobilized in the network of the cytoskeleton, it binds to an rides on the positive ends to be transported across the cell. The transportation is powered by massive local polymerization of actin. The polymerization is also used to push listeria through the plasma membrane into the next cell. Listeria is a food borne pathogen that causes gastrointestinal symptoms.

Question 35

How does listeria move around the cell?

Answer 35

Listeria has a surface protein called ActA, which has an actin binding site and an acidic region which activates the Arp 2/3 complex. When 4 proteins are added (Arp 2/3, cofilin, CapZ, and ATP-G-actin) listeria is able to move. Due to CapZ function, assembly of actin occurs adjacent to the bacteria, where ActA is stimulating the Arp 2/3 complex

Question 36

Cofilin

Answer 36

Cofilin is necessary to accelerate the disassembly of the negative region of the actin filament, which regenerates free actin to keep the polymerization cycle going

Question 37

ARP 2/3 dependent actin assembly during endocytosis

Answer 37

Studied using endosomes that had taken up fluorescently labeled transferrin. Endocytosis assembly factors recruit NPFs that activate the ARP 2/3 complex. Driven by F-actin like listeria. A burst of ARP 2/3 actin assembly drives the movement of endocytic vesicles away from the plasma membrane

Question 38

Phagocytosis and actin dynamics

Answer 38

Actin assembly and contraction drives the phagocytosis of particles. When a bacterium invades, it is coated by antibodies specific to a cell-surface protein in a process called opsonization.

Question 39

Opsonization steps (4)

Answer 39

1. As the fab region of the antibody interacts with the antigen, another domain on the antibody (the Fc domain) is exposed. 2. The Fc domain of the antibody is displayed on the surface of the bacteria it's bound to. It is recognized by the Fc receptor on the leukocyte surface 3. This interaction tells the cell to assemble a contractile actin structure that results in the bacteria being engulfed 4. The bacteria is internalized into a phagosome and is killed and degraded by lysosomes

Question 40

Lamellipodia

Answer 40

Membrane protrusions located at the front of the cell. They play a role in cell motility and are made of cytoskeletal actin proteins.

Question 41

Substratum

Answer 41

A surface that a cell attaches to when moving or growing

Question 42

Steps in cell locomotion (4)

Answer 42

1. Lamellipodia extend from the leading edge (front) of the cell 2. Lamellipodia adhere to the substratum by focal adhesions 3. The rear part of the cell contracts, causing the majority of the cytoplasm to move forward 4. The trailing edge of the cell remains attached to the substratum, but it will eventually retract into the cell body. The membranes and integrins at the back of the cell are internalized and recycled to make new adhesions

Question 43

Which actin based structures are involved in cell locomotion? (4)

Answer 43

1. There is a network of actin filaments in the leading edge that moves the cell forward 2. Stress fibers attached to focal adhesions pull up the cell body as rear adhesions are released 3. Focal adhesions involve attaching stress fibers to the ECM using integrins 4. There is a dynamic actin network in the leading edge, nucleated by the ARP 2/3 complex

Question 44

Diaphanous gene

Answer 44

Linked to sensorineural hearing loss. Actin polymerization involves proteins known to interact with diaphanous protein in Drosophila and mouse. It has therefore been speculated that this gene may have a role in the regulation of actin polymerization in hair cells of the inner ear.

Question 45

Structure of intermediate filaments

Answer 45

IF proteins forms parallel, coiled dimers. It has globular heads and tails that are variable in length. Tetramers are formed by two antiparallel, staggered dimers.

Question 46

Assembly of intermediate filaments

Answer 46

A mature filament consists of 4 protofibrils (groups of tetramers). Globular domains form beaded clusters on the surface.

Question 47

5 types of intermediate filaments

Answer 47

1. Acidic keratins 2. Basic keratins 3. Desmin 4. Neurofilaments 5. Lamins

Question 48

Keratins

Answer 48

There are acidic and basic keratins, which are found in epithelial cells. There are also hard keratins that make up hair and nails. They provide tissue strength and integrity

Question 49

Soft keratins

Answer 49

The type of keratin found in epithelial cells. They make up the skin. The basal layer of the skin cells is in contact with the basal lamina and proliferates constantly, making keratinocytes. Keratinocytes differentiate to make cytokeratins as they leave the basal layer. Cytokeratins make attachment sites between cells to help them withstand abrasion. When the cells die, they leave a cytokeratin network without organelles- the network protects against water evaporation.

Question 50

Desmin, GFAP, and vimentin

Answer 50

Found in muscle (desmin), glial cells (GFAP), and mesenchymal cells (vimentin). Function- sarcomere organization and integrity

Question 51

Desmin

Answer 51

In smooth muscle, desmin filaments link cytoplasmic dense bodies to the plasma membrane so the cells don't overstretch. In skeletal muscle, a lattice of desmin filaments surrounds the sarcomere. Desmin plays a structural role in maintaining the integrity of the muscle

Question 52

Neurofilaments

Answer 52

NFL, NFM, and NFH make up the neurofilaments found in the axons of neurons. They establish the correct diameter of axons, which determines the rate at which action potentials move down the axon

Question 53

Lamins

Answer 53

Most widespread of the intermediate filament proteins. They are components of the network called the nuclear lamina (between the nuclear envelope and the chromatin of the nucleus). There are 3 genes that encode lamins- A, B, and C.

Question 54

Epidermolysis bullosa

Answer 54

Keratin associates with desmosomes and hemidesmosomes, to link cells together or to link cells to the extracellular matrix. Therefore, keratin gives structural integrity to epithelial tissue. In epidermolysis bullosa, a mutation in a keratin gene (K14) results in fragile basal cells that can be damaged just from regular movement. The damaged basal cells cause the more superficial layers of the epidermis to blister.

Question 55

Ortec

Answer 55

Founded in 1991, was formed to commercialize the technology developed by Dr. Mark Eisenberg, an Australian general practitioner, to treat his son for Epidermolysis Bullosa (EB). To prevent his son from having to undergo additional surgeries, Dr. Eisenberg discovered that he could create a substitute skin using cells from infant foreskins and delivering these in a collagen matrix. In 1987, Dr. Eisenberg successfully applied the technology he discovered, now known as OrCel®, on his son

Question 56

Nuclear lamina

Answer 56

Two dimensional mesh that is located between the nuclear envelope and the chromatin of the nucleus. Made up of lamins. LINC complexes attach lamins to the cytoskeleton through 2 nuclear membranes.

Question 57

Intermediate filament associated proteins

Answer 57

The plakin family is one example, which attaches intermediate filaments to other structures. They also link keratin filaments to desmosomes and hemidesmosomes. Plectin is a plakin that connects intermediate filaments to microtubules

Question 58

Desmosomes

Answer 58

Junctions between epithelial cells that provide stability to a tissue

Question 59

Hemidesmosomes

Answer 59

Located at regions of the plasma membrane where intermediate filaments are linked to the ECM

Question 60

MTOCs

Answer 60

Microtubule organizing centers- this is where microtubules are assembled. The MOTC is called a centrosome in an interphase cell and spindle poles in mitotic cells.

Question 61

Protofilaments

Answer 61

Microtubules are made up 13 longitudinal repeating units called protofilaments. In tubulin, the dimers are aligned into protofilaments, which form the wall of the microtubulin.

Question 62

Singlet, doublet, and triplet microtubules

Answer 62

A singlet microtubule is most typical, and is a tube made of 13 protofilaments, found in cytoplasm. In a doublet, there are 10 additional protofilaments forming another tube, found in cilia and flagella. In a triplet, a tubule of a doublet microtubule forms another tubule, making a triplet structure. These are found in basal bodies and centrioles

Question 63

Dynamic instability

Answer 63

Most microtubules will disassemble at 4C and reassemble when cells are warmed to 37C. This method was used to isolate microtubule associated proteins and study their behavior. Microtubules also undergo periods of growth and shrinkage.

Question 64

How does microtubule assembly occur?

Answer 64

The alpha beta tubulin concentration must be above the critical concentration. Once the tubulin concentrations are higher than the critical concentration, tubulin dimers are added to the positive end more quickly than the negative end. The positive end is where beta tubulin is exposed.

Question 65

Graphical representation of microtubule assembly

Answer 65

When tubulin is warmed to induce polymerization, nucleation, elongation, and steady states (level slope) are seen. When individual microtubules in an assembling population have their lengths plotted at different times, we can see them grow and shrink, demonstrating dynamic instability. The graph looks more jagged in this case. Assembly and disassembly proceed at uniform rates, but shortening of a microtubule is much more rapid than growth

Question 66

Dynamic instability depends on

Answer 66

The presence of a GTP beta tubulin cap. The end of a growing microtubule is blunt, while the end of a shrinking microtubule curls like horns. A microtubule with GTP beta tubulin dimers at the end of its protofilaments is strongly favored to grow, but these microtubules also form highly curved protofilaments and will undergo rapid disassembly. The microtubule will switch between growing and shrinking phases (rescues and catastrophes), causing dynamic instability

Question 67

Microtubule pigment cell experiments

Answer 67

Studied using fish or frog melanophores. Melanophores are used to change skin color of fishes and frogs to help with camouflage and social interaction. The changes are mediated by neurotransmitters, and the movement of melanosomes are mediated by changes in intracellular cAMP and is dependent on microtubules. Studies have demonstrated that melanosome dispersion requires kinesin-2 and melanosome aggregation requires cytoplasmic dynein-dynactin. Dynactin might coordinate the activity of these 2 motors

Question 68

Melanophores

Answer 68

Skin cells that contain hundreds of melanin filled pigment granules called melanosomes. When melanophores have dispersed melanosomes, the skin is darker, when the melanosomes are aggregated at the cell center, the skin is paler.

Question 69

Microtubule-associated proteins

Answer 69

Associated with tubulin and help mediate the assembly, dynamics, and function of microtubules

Question 70

Tau

Answer 70

Helps to stabilize the cytoskeleton of neurons. It is a microtubule associated protein. In Alzheimer's and Parkinson's Disease, tau proteins aggregate inside the neurons and interfere with brain function

Question 71

Kinesin-13

Answer 71

Kinesins are motor proteins that mediate transport along microtubules. However, kinesin-13 is not motile. It destabilizes microtubule ends using ATP hydrolysis

Question 72

Stathmin

Answer 72

Proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers.

Question 73

Katanin

Answer 73

Microtubule associated protein that forms a 6 membered ring that severs a microtubule by pulling subunits out of the microtubule- results in its destabilization and severing. This causes more rapid depolymerization of the microtubule, or the microtubule is repaired with GTP tubulin to make a new growing end. It severs longer microtubules during neuronal development

Question 74

Microtubule polarity

Answer 74

Tubulin dimers in a protofilament are oriented the same way, so each filament has an alpha subunit on one end and a beta subunit at the other, demonstrating intrinsic polarity. Subunits are added at the beta end. All laterally associated protofilaments have the same polarity, so the microtubule has an overall polarity.

Question 75

How is the rate of axonal transport determined in vivo?

Answer 75

Through radiolabeling and gel electrophoresis. Radioactive amino acids are injected into the dorsal root ganglia of the sciatic nerve, where the cell bodies of the nerve are located. The amino acids are incorporated into new proteins, which travel down the axon to the synapse. The sciatic nerve is cut to see how far the proteins have been transported.

Question 76

DIC microscopy of vesicle transport

Answer 76

DIC microscopy demonstrates microtubule-based vesicle transport in vivo. An ATP buffer was added to the cytoplasm from a squid axon. DIC microscopy showed that two organelles attached to microtubules move in opposite directions along the same filament, pass each other, and then continue in their original direction. Showed that there must be ATP dependent motors that move cargo along microtubules in anterograde and retrograde directions

Question 77

Microtubule dyneins and kinesins

Answer 77

Dyneins in the cytoplasm mediate retrograde transport of organelles toward the negative end of the microtubule. Kinesins mediate anterograde transport toward the positive ends. Most organelles have one or more microtubule based motors

Question 78

Colchicine

Answer 78

Binds tubulin dimers so they can’t form microtubules. It is able to treat gout because it reduces the microtubule dynamics of white blood cells. Therefore, the white blood cells are unable to migrate and cause inflammation

Question 79

Taxol

Answer 79

Binds microtubules and stabilizes them against depolymerization. This inhibits mitosis by disrupting the mitotic spindle, so Taxol can be used to treat some cancers. It triggers dividing cells to commit to apoptosis, and it blocks Bcl-2.

Question 80

Kinesin-1 catalyzed vesicle transport

Answer 80

Kinesin-1 is located outside of the microtubule and is attached to receptors on the vesicle surface. It transports the vesicles from the negative to positive end of a microtubule. ATP is required for movement

Question 81

Structure and function of the kinesin superfamily

Answer 81

Some of these proteins are involved in the transport of organelles, mRNA, and chromosomes, as well as microtubule sliding and microtubule depolymerization. In these proteins, the conserved motor domain is fused to a variety of class-specific nonmotor domains

Question 82

How does kinesin-1 walk down the microtubule?

Answer 82

This process uses ATP. After kinesin has taken a step, the leading head is in the nucleotide-free state and tightly bound to the microtubule, while the trailing head is weakly bound to the microtubule. The leading head then binds ATP, which causes the region linking the kinesin heads to swing forward and dock in its associated head domain. This thrusts the trailing head forward. The new leading head finds a binding site to weakly bind. The leading head releases ATP, and binds tightly to the microtubule. The trailing head hydrolyzes ATP to make ADP and Pi, and releases Pi. The trailing head is converted to a weak binding state

Question 83

How is kinesin-1 regulated?

Answer 83

The head of Kinesin-1 folds back and interacts with the tail. This interaction inhibits the ATPase activity of kinesin-1. When the motor encounters an appropriate receptor, it unfolds and transports the cargo toward the positive end of the microtubule.

Question 84

The power stroke of dynein

Answer 84

Dynein can't mediate cargo transport by itself. It requires a large protein complex (dynactin) to link dynein to its cargo. Upon ATP binding, dynein dissociates from the microtubule and the linker becomes bent. The linker crosses between the second and third AAA ATPase repeats (pre-stroke). Interaction with the microtubule, hydrolysis of ATP, and release of Pi causes the linker to straighten. The straightening is the power stroke that moves the cargo toward the negative end of the microtubule.

Question 85

Kartagener’s Syndrome

Answer 85

Ciliary dyskinesia, a defect in the dynein arms in cilia. It causes thick mucus that blocks the respiratory tract