Week 8: Cytoskeleton Actin

Question 1

why do cells need a cytoskeleton? (2)

Answer 1

1. provide and maintain shape
2. combine tension and compression

Question 2

what are cells with and without a cytoskeleton?

Answer 2

liquid with a membrane and the cytoskeleton is what makes it spherical or shaped otherwise

Question 3

what would surface tension do to a cytoskeleton-less cell?

Answer 3

Surface tension would carve it into a sphere but structural elements are what mold the cell to how we want them to look

Question 4

what did Wang, Butler and Ingber do as an experiment?

Answer 4

used a magnet to twist beads attached to integrins
- the cytoskeleton will resist the twist when pre-stressed
- ion particles are placed on the cell and a magnetometer will cause them to orient toward the field which creates a twist

Question 5

what did Wang, Butler, and Ingber find?

Answer 5

If you release the twist and then apply the magnet over and over again you can see that there is pretension
- If it was only liquid then it would go back and forth no problem
- The cytoskeleton actively resists the stress so you can see that with the cell

Question 6

what microscopy type lets you look at actin fibers making connections to magnetic beads?

Answer 6

SEM

Question 7

how do cell use the cytoskeleton? (2)

Answer 7

1. helps with molecular motors
2. actively remodeling the cytoskeleton

Question 8

what does the cytoskeleton do for cells?

Answer 8

generates and maintains cell shape and protects against mechanical stress ⇒ wouldn’t be possible if they were only lipid droplets

Question 9

how does the cytoskeleton help cells with molecular motors? (2)

Answer 9

1. organizes compartments so organelles have spatial regions they are placed in
2. transports materials so the cytoskeleton acts like a track to move materials from one side of the cell to another side
   Note: this would imply its a fixed entity and that when it is made it stays that way

Question 10

how does remodeling the cytoskeleton affect cells? (2)

Answer 10

1. they migrate through an environment
2. they respond to intracellular and extracellular signals

Question 11

3 types of cytoskeleton

Answer 11

• intermediate
• actin (smallest)
• microtubules (largest)
  → the cytoskeleton is a polymer and has filaments
• How the filament is organized gives a clue to which one it is

Question 12

visual properties of intermediate filaments

Answer 12

like spaghetti with a meatball nucleus

Question 13

visual properties of actin filaments

Answer 13

stays on the periphery of the cell

Question 14

visual properties of microtubule filaments

Answer 14

spider like appearance from MTOC from a central location of the cell

Question 15

what do intermediate filaments do in epithelial cells?

Answer 15

connect cells together which allows for a continuous epithelial sheet
- Epithelial cells need to maintain a barrier so things on the outside cannot come in
- Things would come in and invade the body

Question 16

what do microtubules do in epithelial cels?

Answer 16

make up the longer distances from one end to another (apical to basal)

Question 17

actin

Answer 17

building block of cytoskeleton which is a polymer built from monomers

Question 18

what type of protein is actin?

Answer 18

globular protein (globe like)
- it has alpha helices and beta sheets and a nucleotide referring to ATP or GTP
- the protein is not symmetric and when looked at from different sides it wont present the same (boxing glove look)

Question 19

what is the role of ATP in actin?

Answer 19

at the core of actin is an ATP molecule
- Actin is an ATPase which means it hydrolyzes ATP
- goes from a Triphosphate to diphosphate making it ADP when in the filament

Question 20

how do actin filaments connect?

Answer 20

One is placed on top of the other and it will only connect in one spot ⇒ with a slight angle which causes the rotation of the filament
- This creates the alpha helix of the filament ⇒ slowly turns and can be followed
- The helix is a double helix ⇒ eventually results in one unit the same orientation of the helix

Question 21

pinch of the helix; myosin connection?

Answer 21

the next monomer that has the same orientation of the first one ⇒ 37 nanometers
- Myosin hops along the filament and where it binds depends on where the next monomer is that has the same orientation

Question 22

distance between actin monomers in the same orientation?

Answer 22

about 36.4 mn ⇒ 2.8 x 13 monomers

Question 23

describe the polarity of actin filaments

Answer 23

top and bottom of the actin filaments are different
+ end: add actin
- end: lose actin

Question 24

what does the myosin protein structure look like?

Answer 24

has a barbed (plus) end and pointed (minus) end
- When you put myosin on the filament you can see arrow headlike shapes showing the direction of movement

Question 25

another phrase for the plus end of actin?

Answer 25

barbed end

Question 26

another phrase for the minus end of actin?

Answer 26

pointed end

Question 27

tread milling

Answer 27

filament length stays the same ⇒ analogy for the flexing of the filament - Looks like it isn't changing but it is, but it doesnt change length of the filament

Question 28

what happens when you mix in salt with actin monomers?

Answer 28

when actin monomers are mixed with salt (implies there is no salt at the beginning of the experiment) in a test tube over time the % of actin subunits increases after salt addition - You can measure turbidity of the solution because polymers scatter light - makes an S curve where the beginning is exponential growth and then tapers off

Question 29

explain the stages after salt is added to actin monomers? (3)

Answer 29

1. nucleation where the lag occurs when it takes multiple actin monomers before we can start taking some off again 2. elongation (exponential growth) where once a nucleus is formed (building blocks) then you can take some off of it again but actin filaments grow fast 3. steady state equilibrium phase where treadmilling is occurring - things add and subtract at the same rate

Question 30

what does adding seeds do to the nucleation phase of actin elongation?

Answer 30

- Will speed up the production of the actin filament building - Without the seeds it will be a random process ⇒ depending on where you put the seeds, you can organize the membrane more - Looks much more linear and doesn't have a nucleation lag phase

Question 31

seeds definition

Answer 31

preformed packets of some actin filaments

Question 32

why doesn't the actin subunit filament curve reach 100%?

Answer 32

because there are some free subunits needed for addition of nuclei while others are being taken off

Question 33

cardiomyocyte

Answer 33

organized/integrated clusters of cells in your heart that expand and contract - millions work together to case a muscle contraction simultaneously

Question 34

muscle

Answer 34

a coordinated actin-myosin interaction ⇒ sliding actin filaments - the myosin has a straw cleaning brush structure

Question 35

thick filaments

Answer 35

braiding of multiple myosin - a lot of tubes together with a head on it all braided together so the heads are staggered at different places - helical pattern and there are long arrays of heads

Question 36

bare zone

Answer 36

place where there is no heads in a muscle fiber - on the myosin filaments

Question 37

components of an actin/myosin muscle filament (6)

Answer 37

- The heads will bounce on and off the filaments - A phosphate is kicked out of the ATP which will cause the lever to flex - Energy from ATP is converted to mechanical motion - The ATP is placed back in and the cycle repeats - The head must detach after each stroke - Because they do this on opposing sides the actin filaments will move inward toward the bare zone

Question 38

how many myosin heads move at a time?

Answer 38

Only 1-2 heads will stroke and come off at a time - this happens at random but in a short enough time span that it is coordinated to move the muscle because many are triggered at nearly the same time

Question 39

T/F myosin heads are detached for most of the cycle?

Answer 39

true

Question 40

where do actin filaments anchor?

Answer 40

Z disks which is the opposite side of the dead zone at the + end side - this is not treadmilling because there is a protein that latches on to the actin on the minus end so it can no longer fall off as well as a plus end capping protein - The two Z discs are getting brought close to one another when a contraction occurs

Question 41

what triggers the contraction of a muscle?

Answer 41

when calcium comes in through gated ion channels - myosin and ATP interact

Question 42

sarcomere

Answer 42

a building block of muscle composed of one Z disk on one side of a unit to the Z disk on the other - basic contractile unit of movement in a muscle fiber - there are multiple myosin filaments lined up on the same Z disks

Question 43

myofibril

Answer 43

large structure of sarcomeres (make up a circular unit) - these are stacked on top of one another and make circular tubes which are lined up next to one another to make a muscle fiber - there are nucleus' spread throughout them and are multinucleate cells so that you cannot tell when one cell begins and another ends

Question 44

sarcoplasmic reticulum

Answer 44

structures that surround each of the myofibrils - includes transvere (T) tubules

Question 45

what is a transverse (T) tubule?

Answer 45

formed from invaginations of plasma membrane around the sarcoplasmic reticulum and myofibril

Question 46

what happens when electrical impulses depolarize muscle cell membranes?

Answer 46

voltage gated calcium channels will open and calcium floods in to the cell - As long as resting membrane potential is there the voltage gate will work - When open the calcium floods out of the transverse tubules and create a calcium induced calcium release - These lead to a dump of calcium coming from the sarcoplasmic reticulum which leads to the contraction

Question 47

what are the 2 things layered on actin filaments?

Answer 47

1. tropomyosin 2. troponin - These block the sites where myosin will bind to the actin filaments - When calcium comes in the tropomyosin twists so myosin binding sites are exposed

Question 48

tropomyosin

Answer 48

long slender protein that sits on top of the boxing gloves in a helical format since it follows the actin binding pitch of helix

Question 49

troponin

Answer 49

3 subunits which forms a complex with tropomyosin to sit on top of the actin

Question 50

when does muscle myosin bind tightly to actin filaments?

Answer 50

low ATP and high calcium - high ATP causes all the heads to bounce more to ATP so it will bounce around trying to find a site to bind ⇒ it binds and latches on, hydrolyzes ATP, and then the lever arm will move

Question 51

2 nodes of the heart

Answer 51

atrial and ventricular - atrium contracts first which fills ventricles which then pump upwards

Question 52

types of muscle (3)

Answer 52

1. skeletal which operates the same way as cardiac but has a different type of myosin (alpha) - Speed of contraction is faster than the speed of contraction for the heart in humans 2. cardiac muscle is slower but consistent contractions in and out pumping motion (beta) 3. smooth muscle which use contractile units but are not organized as sarcomeres - Contract and response to specific stimuli and are organized differently

Question 53

how many individuals have genetic mutations leading to abnormal cardiac function?

Answer 53

1/500 for mutations in several cardiac muscle contractile proteins that can lead to dilated or hypertrophic cardiomyopathy - also may lead to heart failure and premature death

Question 54

idiopathy in heart issues

Answer 54

don't know the cause for the illness ⇒ some of the contractile proteins are not functioning normally due to gene mutations - Tropomyosin, troponin, et.c ⇒ most are beta cardiac mutations - If any part of the cycle isn't correct you will start to get hypertension ⇒ now we have small molecule inhibitors as treatments

Question 55

neutrophils

Answer 55

cells that chase and engulf bacterium to remove them from out body system - first line of defense in the immune system when we have an infection

Question 56

how do cells use actin to move?

Question 57

types of actin networks for movement (4)

Answer 57

1. stress fiber ⇒ contractile bundle - Looser bundles that are antiparallel - Can create stress 2. Cell cortex ⇒ gel like network 3. Lamellipodium ⇒ dendritic network 4. Filopodium ⇒ tight parallel bundle Projections sticking out like antennae

Question 58

types of tight vs loose bundling proteins in actin?

Answer 58

- fimbrin is a tight bundling protein - alpha actinin is a loose bundling protein

Question 59

components of actin filament branching networks

Answer 59

ARP2/3 complex which looks similar to actin monomers - Nucleation promoting factors will mimic an actin nucleus and allow the ARP2/3 complex to let actin filaments add to the plus end - The minus end is stabilized so depolymerization no longer occurs and the actin can keep growing ⇒ creates the actin filament on demand which allows actin filament to be created anywhere in the cell - sit on an existing filament and the angle that it sits on allows it to grow at a certain angle of 70 degrees ⇒ this allows for numerous amounts of branching to create a dendritic (tree like) actin network - composed of a mother (actin) and daughter (ARP2/3 start) filament

Question 60

how do ARP2 and ARP3 work together?

Answer 60

these units bind together to form a complex which is then nucleated by a nucleation promoting factor (NFP) - actin filaments are added to the plus end and the complex is at the minus end

Question 61

types of cap filaments

Answer 61

uncapped where there is growth at the plus and minus ends vs capped where there is growth at the one end only

Question 62

what type of cap is ARP2/3 complex?

Answer 62

this caps the minus end of actin so the plus end can grow

Question 63

in the graph from class what did the red and green like represent?

Answer 63

red line is without a capping protein which requires higher concentrations of actin filaments to grow the actin whereas the green line

Question 64

off vs on rate for actin filament

Answer 64

- off rate is first order so it is a constant - on rate is second order and dependent on concentration so this will determine if actin grows or not

Question 65

cofilin

Answer 65

protein that strains actin filaments by changing helical pitch ⇒ cofilin binds ADP actin (old actin filaments) - When it goes on the actin it creates a twist (strain) by reducing the pitch of the helix (74 to 57) - The actin filament does not like the strain so it wont stay this way for long and will collapse after time ⇒ now this actin can be used again

Question 66

how does migration work?

Answer 66

dendritic network forms and cofilin hops on to take them apart which allows for new actin monomers to diffuse to the front and let barbed ends to grow - Growth is always going up because barbed ends are on the top

Question 67

what is the relationship between cofilin and ATP/ADP

Answer 67

Cofilin binds ADP actin which means that the actin site for ATP has been hydrolyzed from being polymerized for some time already - binds at the back because thats where the old actin is - ADP will come off and ATP will come back on in the cytosol which activates the new actin filaments

Question 68

which side of the cell are actin and cofilin found?

Answer 68

the leading edge side

Question 69

focal adhesion contacts

Answer 69

divots in the cell base where the cell is attaching to the basal lamina via integrins ⇒ the cell moves by putting contacts down one at a time while the lamellipodium pushes forward - Actin and binding proteins drive protrusions - the cell creates the lamellipodium by the branch network - As the branch forms in the front it causes the cell membrane to push forward