Notes 3: Cell Motility and Contraction

Components of the Eukaryotic Cytoskeleton

• in order for us to talk about cell mobility and cell contraction we need to introduce the third class of cytoskeletal projects which is the actin filaments

Actin filaments regulate cell shape

• actin is a marvelous project involved in a lot of different things

• it's present in for example the microvilli which are this extensions of your intestinal cells that effectively capture the nutrients

• they are in muscle cells and myofibroblast and the actin filaments involved in the contraction of the cells

• they are at the leading edge of moving cells

• They even form the circular rings during cell division which are very temporary and that basically strangle the cell during cell division

Actin Structure and Assembly

• Right OK in a way the actin filaments may remind you of the microtubules but they're very different

• why are they different

• well they're different in three main aspects

• one the actin filaments are just they they don't make the tube structure like the microtubules, each filament is just one filament OK and each filament is composed of these dimers OK the actin dimers that wrap around in a helix like this

• And that's just it

• you don't get like in the microtubule you have the tubulin dimers that makes the protofilaments and then you need 13 protofilaments to make the microtubule

• not in actin, there's no protofilaments and filaments are just one thing and that's the filament and that's the actin filament that's the difference number one

• difference #2 is that the dimers are ATPases, they're not GTPases

• OK so the nucleotides that provides energy in the in actin polymerization is not GTP is ATP

• So GTP for microtubules, ATP for actin

• to remember because actin ATP microtubule GTP

• the third difference is that the microtubules only grow towards the plus end, grow and shrink at the same place, whereas the actin filament grow towards the plus end and shrink towards the minus end

• so effectively there's the directionality

• they grow here and shrink here grow here and shrink here because actually there's a fourth difference

• the actin filaments they're not anchored to a center like the microtubules the microtubules are anchored to the centrosome and grow towards the opposite end which is called the plus end whereas the actin filaments are not anchored to anything, they grow at the plus end and shrink at the minus end which is the opposite end

Cycles of cortical actin polymerization and myosin-mediated contraction allow cell movement and migration “forward”

• OK this is basically how cell movement works

• there are a lot of you know different scenarios and there's it's a lot more complicated than this in real life

• but basically what happens is that there's a leading edge which is formed by cortical actin

• by cortical actin we mean the actin that's underneath the the plasma membrane and that forms like this cortex all around this cell like envelopes the cell

• OK that's cortical actin

• so at some point there's what we call the leading edge of the cell which is a point of the cell where the mesh of cortical actin grows towards that end in that direction and stretches the cell

• OK so this is the cell and towards what we call the leading edge it becomes a leading edge because the actin the cortical actin is growing and it's causing this tension

• You can feel the tension

• what happens in the other end is that two things happen

• actin will start to depolymerize and myosin at this end will contract

• so as I'm here leading edge goes that way and stretches the cell that way

• now the actin that polymerizes and also the myosin contracts

• so I go like this and like this, kind of like a worm

• and that's how cells move

• Leading edge forms the lamellipodium that is attached to a substrate through cell matrix interaction

• I think you might have covered this with professor Martin the main cell matrix interacting proteins are the integrins which are heterodimers that require calcium to interact with the extracellular matrix

• Then there are many other different types of cell matrix interaction

• but really the integrins are the main

• so that lamellipodium is attached the cortical actin grows and detaches and then attaches again and there's contraction at the opposite end that moves the cell forward as the new attachment forms and then more lamellipodium forms detaching and then it starts over again, it attaches and then contraction and the cell moves

Actin-related proteins (ARPs)

• And this is keratocyte moving really beautiful if I can make it start

• this is the cortical actin I was talking about

• this is an electron micrograph which shows this mesh of cortical actin

• This video sequence shows a single fish skin cell moving across the field of view

• the cell body containing the nucleus and all the membranous organelles is at the left side

• the large broad flat lamellipodium that pulls the cell forward to the right is filled with a dense network of actin filaments

• so in this diagram you can see how at the leading edge the plus end of the actin filament is stabilized by the capping proteins which are not attached at the minus end where the actin shrinks

• here it's stabilized and here it's destabilized and it shrinks

• so here it grows here it shrink

Myosins are Motor Proteins

• the myosins are also motor proteins

• just like the other motor proteins, have a globular head and I believe a linear tail

• they usually exist as dimers

• again in this dimers the tails dimerize

• the head they don't necessarily dimerize they work as a monomer

• the head of the myosins always interact with actin and the tail interacts with whatever cargo

• so the movement is towards the plus end because the myosin head always moves towards the plus end of the actin right

Muscle-specific myosins have evolved special structural organization

• This is when it dimerizes

• when it dimerizes

• myosins are always dimerized in forming the thick filaments of skeletal muscle cells and also cardiac muscle cells, contracting muscle cells

• so the way they work is there's this dimer of the myosin with the dimerized tail and the two heads and several dimers organize themselves tail to tail

• you can see it on the picture but the point is that they organize themselves this is a dimer here coiled-coil with a head and then another one sticks on top like this and then another one like this

• there's a bunch of them that make a bundle

• then on the other end there's another bunch that goes like that OK

• So what you have is this strong structure that you can see in the picture where there's in the middle there's just the two ends of the tails that are going like this

• but then there are this, these other two right and left big structures with a lot of myosin heads

• OK and they're all around like a like a tubular structure

• and then the actin is gonna go all around this well

Anatomical structure of muscle cells and the contractile unit: the sarcomere

• So here in in red is the thin filament, the actin

• in green the myosin

• here at this interface where there's both green and red is where the heads of the myosin are, interacting with actin

• and this is the Z disc that keeps the thin filaments from one Z disc to the other Z disc is called the sarcomere, and keeps the sarcomere together

• so um it basically binds the minus ends of the actins together so it's just sort of capping it, it contains kind of a capping protein in a way

• the main protein that is in here in the Z disc that keeps together the sarcomeres is called Titan

• it's an enormous ginormous protein is the biggest protein of the body 700 kilodalton

• so the striation of the striated muscle it comes from that

• this is the dark part is where there's myosin

• the very dark part is where there's both myosin and actin

• the light part is where there's only actin because the actin filaments are thin whereas the myosin filaments are fairly thick because there's a lot of myosins stacked like that

• then the Z disc is also pretty dark because there's all the proteins that are keeping together the 2 minus ends of the of the actins

Muscle Contraction

• This is how muscle contraction works

• Neuron stimulates a muscle cell

• an action potential sweeps over the plasma membrane of the muscle cell

• The action potential releases internal stores of calcium that flow through the muscle cell and trigger a contraction

• muscle cells have an elaborate architecture that allows them to distribute calcium ions quickly throughout the cytosol

• deep tubular invaginations of the membrane called t-tubules crisscross the cell

• when the cell is stimulated a wave of depolarization that is an action potential spreads from the synapse over the plasma membrane and via the T tubules deep into the cell

• a voltage sensitive protein in these membranes opens a calcium release channel in the adjacent sarcoplasmic reticulum which is the major calcium store in muscle cells thereby releasing a burst of calcium ions all throughout the cytosol of the cell

• within a contractile bundle of muscle cell called a myofibril the calcium interacts with protein filaments to trigger contraction

• in each contracting unit or sarcomere thin actin and thick myosin filaments are juxtaposed but cannot interact in the absence of calcium

• this is because myosin binding sites on the actin filaments are all covered by a rod shaped protein called tropomyosin

• a calcium sensitive complex called troponin is attached to the end of each tropomyosin molecule

• when calcium floods the cell troponin binds to it moving tropomyosin off the myosin binding sites

• Opening the myosin binding site on the actin filaments allows the myosin motors to crawl along the actin resulting in a contraction of the muscle fiber

• calcium is then quickly returned to the sarcoplasmic reticulum by the action of a calcium pump

• without calcium myosin releases actin and the filaments slide back to their original position

Muscle Contraction Steps

• individual steps

• once get calcium and myosin can move

• so starting from when the tropomyosin is going away

• Troponin is bound to calcium undergoes a conformational change that moves away the whole complex

• tropomyosin is away now actin and myosin can interact

• OK the myosin head is an ATPase so ATP comes in that's why you need energy for your exercise

• ATP binds to the myosin head and now the myosin head is bound to ATP

• see the position of the myosin heads OK now it's like straight

• ATP hydrolysis follows and the myosin head moves a little bit like this and it's now bound to ADP and phosphate

• OK releases phosphate and bound to ADP

• now binds to actin

• so it started off close to this actually bound let's say to the dimer of actin

• then it binds ATP detaches, so it's bound here binds ATP detaches, hydrolysis, moves, releases phosphate, attaches again with ADP bound and then releases ADP and moves straight again

• then the cycle starts again ATP detaches to do

• so this movement as you can see allows it to slide in this direction along the actin filament

• and if you remember what was in the video there are all these myosins sliding and crawling along the actin filament

• and because the structure of the thick filament is this way these are crawling in One Direction these are crawling in the other direction and the actin filaments are not going anywhere they're not polarizing or depolymerizing because they're kept within the Z disc

• so what happens is that effectively in order for this movement to happen the whole thing shrinks

• they slide onto the actin and the sarcomere shrinks

• and then when there's no more calcium, it returns

• contraction, relaxation, contraction, relaxation

• that's how you move, you breathe, see, control blood pressure

• Muscle does a lot of things