ZOO 138, Wednesday, January 15, 1997, 12:00 p.m.
Any questions so far about anything I have said or anythingthat has happened in lab or anything that happened in a previouslecture?
We're getting behind here because we're supposed to be dealingwith muscle today, but I want to finish off talking about neuromuscular junction, which is a place where a nerve cell contactsa muscle cell, and where the information which is traveling orbe propagated along the axon of a nerve cell or a neuron -- that'sa synonym for neuron. Where the information is being propagatedalong the axon reaches the muscle cell.
And one of the most important things that you will hear andsee over and over again in biology is the idea that form followsfunction. That you to -- if you can deduce certain things aboutwhat's going on if you understand anatomy, and conversely youreally cannot understand how something is working unless you understandthe anatomy, that arrangement of the structures.
So I'm going to start with a diagram here. And we're going toexpand on this diagram, probably won't get a chance to completelyexpand upon until I make up for the material that I don't getto today on Friday.
So this is an axon. I have sort of reoriented that diagram ofan axon by 90 degrees. This is the axon and this is the axonterminal or synaptic terminal expanded part at the end of oneof those branches that you see out of the right end of the thing.This is the synaptic cleft, it's the space between the nerve celland the muscle cell, a couple hundred angstroms across.
Then this black line here is the muscle cell membrane, whichis called a sarcolemma. The muscle cell itself is really hugecompared with the axon terminal. This muscle cell would go --you know, from this point right here, it would go down a coupleof floors and it goes off in that direction to the east, you know,a hundred yards or something like that. It's just a tiny partof a really huge cell that we're drawing here.
In the axon terminal we're going to find some little spheresof membranes. Little bubbles of phospholipid bilayer, hundredsof them actually, not just is a small number like this. So thisis a diagramatic representation. And these are called "synapticvesicles."
And this word "synapse" over "synaptic"is a term that comes from nerve anatomy. A synapse is a pointwhere another nerve cell contacts another nerve cell, but theanatomy is the same. And those synapses between nerve cells arevery, very small and hard to study. And most of what we know abouthow synapses function is actually based upon the study of theneuro muscular junction, which is what I'm diagramming here.
These synaptic vesicles contain a single chemical. Which inthe case of most of the skeletal muscle of vertebrates, this chemicalis called acetylcholine. And acetylcholine is, if we look at synapsesin the nervous system where nerve cells contact nerve cells, whatwe will see is very a similar arrangement of an enlargement atthe end of the one nerve cell with synaptic vesicles in it andthe chemical contained within the synaptic vesicles, but it'snot always acetylcholine.
It might be a chemical called Noradrenaline or Norepinephrineor Gabaminobuteric acid or GABBA (spelling). And there is dozensand dozens of chemicals that are found in synaptic vesicles indifferent parts of the nerves system, and they are the called"neuro transmitters." And acetylcholine is a neuro transmitter.
So even though this is in a muscle cell or in a nerve cell,it's going to communicate with a muscle cell, so-called a neurotransmitter.
Now, we're going to find the sarcolemma, underneath the synaptic-- the axon terminal, we're going to find 3 membrane-bound proteins.
So these are floating in the membrane here. And there arelots of them, not just one of each type. I'm just diagramaticallyrepresenting, though, one of each type.
And the first one I'm going to mention is a receptor molecule,which is the acetylcholine receptor.
Now, the abbreviation that we use for acetylcholine is ACH.This is the acetylcholine or the ACH receptor.
One which is an integral part or actually directly associatedwith the ACH receptor is a "sodium channel," a normallyclosed sodium channel.
Now, I'm diagramming these as though they were separate molecules.In fact, they are 2 different parts of the same molecule.
It used to be thought they were separate molecules. And nowit's understood that they are kind of in 2 separate functionalparts of what is molecularly the same molecule. But it's helpfulin terms of understanding the function to keep the idea in mindthat they function differently from one another.
And then the third type of molecule that we find here is a anenzyme. It's called "acetylcholine esterase," or ACHase.
That's the anatomy of the neuro muscular junction, a diagramaticrepresentation. The axon terminal is actually much bigger anit looks sort of like a spider when you look at it from the top.It's a big structure. If you look at it from the top it kind ofbranches and has a bunch of little extensions from it, so thatthe nerve comes down like this, if you can imagine it coming downout of space, and then it has these extensions. It's bigger.
And that's in order to achieve a large area of contact betweenthe nerve cell and the muscle cell. This branching produces anextra area of contact.
Now, what happens when an action potential is propagated downthe axon and that depolarization, that reversal of polarity, thespike phase, and that hyperpolarization phase, when that getsdown the axon terminal it causes a couple of a small number of,and a fairly consistent number, of these synaptic vesicles toundergo a process called "exocytosis" where they fusewith the membrane at the end of the cell and dump the acetylcholineout into the synaptic cleft.
That process is called "exocytosis."
It's like if you have studied basic cell biology, you know howa water vacual dumps water out, it's the opposite of the way inwhich micro organisms ingest foods, think about how an amoebaeats or how they invaginate and intake. So it's the exact opposite.
Because the synaptic vesicle is formed of a phospholipid bilayer,the same material as the membrane, it fuses with the membraneand it actually physically becomes part of that part of the membrane.And the stuff, the acetylcholine that was inside the synapticvesicle now is outside of the cell, and it's in the synaptic cleft.
It's going to diffuse across the cleft by random molecular movement.It's going to encounter -- some of them are going to encounterthe acetylcholine receptor.
And when the acetylcholine combines with the acetylcholine receptorit causes -- through a molecular rearrangment, it the causes thesodium channel to open.
That normally sodium closed sodium channel opens, and sodiumwhich is at a high concentration in the fluid surrounding thecell will diffuse through the sodium channel into the interiorof the cell.
Just as sodium diffuses into the nerve cell in order to makean action potential, exactly the same thing happens, lots of sodiumchannels are associated with acetylcholine receptors and a largeamount of acetylcholine gets dumped out and so a bunch of acetylcholinereceptors all get have to acetylcholine associated with them.
A whole bunch of sodium channels open up and the membranedepolarizes and produces an action potential in the sarcolemma.So we get the same kind of depolarization of the membrane potential,and that propagates or spreads over the sarcolemma, the musclecell membrane.
And that's really the function of this neuro muscular junction.It's a to take an electrical signal, which is an action potentialin the axon, and causes there to be an electrical signal, an actionpotential in the muscle cell membrane.
So we have a piece of information that is going to cause thismuscle cell to become active, to contract. And that informationis an electrical signal, and it gets transformed very brieflyinto a chemical signal. This is a chemical synapse. So that theinformation is transmitted from one cell to the next cell in theform of a chemical, which is the neuro transmitter.
The acetylcholine is not a huge molecule but its not lipid solubleand it cannot get into the cell. That's why the receptor has tohave its stereo specific site on the outside of the cell. Acetylcholinenever comes into the muscle cell. The only thing that comes inis sodium, depolarized in the membrane and causing a wave of depolarizationto spread outward over this whole entire muscle cell.
We have to be able to turn this signal off. If we wants themuscle to be able to contract and then relax, which we alwaysdo want muscles to do that, then we have to be able to turn thesignal off. And as long as acetylcholine is combined with thereceptor, the sodium channels are going to be open. So we haveto some way to turn off the chemical signal. And that's what theacetylcholine esterase does.
The acetylcholine, when it first -- say, it first combines withthe receptor, it does not stay bound to the receptor, it doesn'tform a covalent bond with the receptor. It kind of bounces inand sits there and the sodium channel opens up and then throughrandom molecular movement, acetylecholine pops out again.
When it pops out, it may bump into another acetylecholine receptorand cause it to open up and a little bit of sodium to come in,or when pops out of the receptor it may bang into acetylcholineesterase, which is going to split acetylcholine into 2 molecules,a choline molecule and an acetate molecule.
So this esterase takes ACH and converts it into choline plusacetate. Now, neither choline nor acetate has the same molecularshape as acetylecholine. And therefore neither one of these, theymight bump into an acetylcholine receptor but they're not goingto look like acetyl choline.
This is the way in which the signal, the chemical signal isturned off by converting it into 2 separate parts, neither oneby itself can stimulate the acetylcholine receptor and cause thesodium channel to open up. So what you get is a blast of acetylcholine,a bunch of the receptors combined with acetylecholine, the sodiumchannel opens up, the membrane depolarizes, and you eat up theacetylcholine itself and turn off the chemical signal.
In fact, what is happening is you have a chemical reaction,you have the receptor, plus acetylcholine reversibly goes to forma receptor ACH complex.
And as with any chemical reaction, the equilibrium positionof the reaction is going to be a function of the concentrationof reactant. So when exocytosis takes place and acetylcholinegets bumped into the cleft, there is a high concentration of reactants.What does that do to the equilibrium position? It shifts it towards-- so what we find is a lot of receptors that have acetylecholineassociated with them.
Now, the acetylecholine esterase starts chewing up the ACH.And eventually the concentration of ACH in this area of the musclebecomes zero. And when this concentration is zero, then the equilibriumposition shifts back. We have receptors that don't have acetylcholineassociate with them, and the sodium channels are closed, and sothe membrane repolarizes. And that cause a muscle cell to undergoa single instantaneous twitch that might last for 50 millisecondsor so. So this whole process happens very very quickly.
STUDENT: What causes the synaptic vessels to close back up?
INSTRUCTOR: They don't close back up; they have to be reformed.You can imagine that as a synaptic vessel fuses with the membrane,the membrane itself actually becomes bigger, right? Due to theaddition. So there has to be a process going on somewhere elsewhere that membrane is being made back in to synaptic vesiclesand then getting filled up with acetylecholine. So the cell iscontinually having to refabricate acetylecholine.
The choline diffuses back into the nerve cell and gets an acetatemolecule put on and a new vesicle gets formed, and it gets filledup with acetylecholine.
STUDENT: But you said that it's not permeable to the membrane.How does it fuse back up into the vesicles?
INSTRUCTOR: Well, there has to be transport mechanism for cholineto get it back in. But, again, somewhere else in the cell we havea process going on where we have new vesicles being formed andthe choline is being taken up by the cell, made back into acetylcholine and packaged back up into more synaptic vesicles. It's not partthe story. It's part of the recovery process.
And there more to it. There is calcium and all kinds of fancystuff that causes exocytosis as well that I'm not telling youabout.
That's how the neuro muscular junction works. We need to talkabout what is going on in terms of the muscle itself before wecan understand how this action potential that we have just producedis going to cause the muscle cell to work.
This is covered in your Illustrated Notes, the structure andfunction of vertebrate skeletal muscles. So I want you to takea look at that part now.
This is what your notes look like.
Now, a skeletal muscle is really a wonderful example of a situationin which there is a hierarchy. That is, there are various levelsof organization. We can start off, we really need to understandthe whole entire hierarchy, starting at the gross anatomical structureof muscle. When we say it's "gross" that means it canbe seen without looking in a microscope.
You know, you have biceps muscle, that is what is shown up here,the whole entire muscle. It's composed of a very large numberof individual muscle cells which are the next level of organization.So any individual muscle in your body can be composed of a differentnumber of muscle cells or muscle fibers as they are called. Somevery small muscles may have only a hundred cells, huge musclesmay have 100,000 cells.
And they are normally attached to a bone by a connective tissue,by a tendon.
Although, there are lots of other arrangements that also occur.
And so this is the level where we are talking about a wholeorgan, a muscle. And now we're starting to sort of expand andenlarge some of these parts of this individual structure. Thesetubular structures here are the skeletal muscle fibers. And that'sa useful point of reference for a biologist is to know where arewe dealing with a whole cell, because we have a set whole setof expectations about a whole cell.
It's going to be surrounded by a single continuous membrane.It's going to be filled with cytoplasm. And the muscle fiber isthe synonym for a muscle cell.
And there are some peculiar things about this cell, for onething it is multi nucleate. It has many nuclei. As you can seein the diagram, each little oval-shaped structure is a nucleous.
How is it possible for a single cell to have many nuclei. Ourstandard cell has only a single nucleous.
Well, the answer is that this cell forms during the embryologyof the animal by a diffusion of a large number of individual embryonicmuscle cells which are called myoblasts. You have probably encounteredthat term "blast" in lab, like, an osteoblast. Well,a "myo" the prefix means muscle. "Blast" isa cell that forms muscle.
So embryonic myoblast, each of them with their own nucleous,fuse to form the single skeletal muscle cell. And that's why thereare any nuclei in there. Each nucleous is active; it actuallycontrols a certain local area of the muscle cell.
So that if you start exercising, for example, and building upmuscle or you the do the opposite you become a couch potato, youstart to get out of shape, your muscles change and all kinds ofthings are changing within the cytoplasm of the muscle cell, andthat's under the direct control of a nucleous in the local area.
So nuclei are not unimportant. They are active, and they areregulating local areas of cytoplasm.
The other thing that's important to know about this muscle cellis that functions as a unit. In other words, when you activatea muscle cell, the whole entire cell goes through the processof contraction. You don't have part of a cell active and anotherpart inactive.
And the reason for that is that the muscle cell is surroundedby a single membrane and this action potential, that we just talkedabout being performed by the neuro muscular junction, that actionpotential is going to spread over the entire membrane of thissingle cell and trigger the action -- trigger the contractionof the cell.
Now, there are a number of organelles, intracellular organelles.That is a little tiny structure inside of the cell that we needto know about in order to understand how this muscle cell functions.And the first of these intracellular organelles we need to knowabout is myofibril.
So there are 2 terms that are very, very similar to one another.Muscle fiber, sometimes called a "myofiber." Those area muscle cell. And "myofibril" is an organelle withina muscle cell.
This ending "fibril" should make you think of somethingthat's a little bit smaller. So that maybe is one way that youcan remember the difference between a myofiber and myofibril.A myofibril is an organelle, a little organ within the musclecell itself.
And these myofibrils are themselves also bundles of a kind oftubular structures. They are sort of tubes within a tube. If youlook at the diagram, you can see that the muscle is composedof a bunch of muscle fibers, they are all kind of tubular. Butif you look inside of a single muscle fiber, you see other tubularstructures, which are the myofibrils. So you have bundles of bundles.
Now, the things that are located within the myofibrils are thefilaments. Also, sometimes called "myofilaments."
So this word "myo" which means muscle gets involvedin all these things. There is another word called "sarco."There are Latin an Greek for things. And "sarco" isGreek, and "myo" is Latin. And they both get used.
Now, the filaments, there are 2 types of filaments. There arethick filaments and thin filaments.
And the thick and thin filaments, we'll talk about in a minute.But they appear a very, very regular fashion, a very regular geometricalarrangement, if you will, of these thick and thin filaments, whichis diagramatically represented here at the bottom.
But bundles of these filaments surrounded by another structurethat I'll be telling you about in a minute, make up a myofibril.And then a whole bunch of myofibrils, surrounded by muscle cellmembrane, make up the cell.
So here are the filaments. Bundles of filaments make up a myofibril.A whole bunch of myofibril surrounded by a muscle cell membranemake up a muscle cell.
Now, the 2 types of filaments are the thin and thick filaments.
The thin filaments are composed of "fillamentous actin"or "F-actin." And you can see several different representationsof what F-actin looks like in your Illustrated Notes here.
They are like 2 helically wound strings of beads. If you canimagine a necklace, a pearl necklace, you pull it out straightand you twisted it, you have a whole bunch of little pearls allsort of strung together in two parallel strands, but they wouldbe wrapped around one another. That's what is represented here.That's a fillamentous actin.
So here is this fillamentous actin. In the analogy of the pearlnecklace, each of these would be an individual pearl.
Those individual pearls are G-active, globular actin. And actinis a protein. We have gone from a level of an organ, the muscle,all the way down to a molecule, a fairly large protein moleculethat is globular more or less spherical in shape. And you takea whole bunch of these globular actins, and they will stringthemselves together into fillamentous actin. So they have placeswhere they can form bonds to one another. And those then makeup these thin filaments.
The other filament, the thick filaments, are composed of anotherprotein called "myosin." And there are some other proteinsassociated with thick filaments as well, but we won't worry aboutthose until later.
The individual myosin molecules have several sub-units. Theyhave long tail section, and they have a globular head that projectsout to the side from the tail section.
And they are 2 important things to remember about the myosinhead, that is the sub-unit that sticks out to the side. It has2 catalytic sites on it. It two chemically reactive areas of thisprotein molecule.
One of those is a site that can form a covalent bond to actin.It's called the actin-binding site. So on the diagram here, thelittle head has 2 little oval dark places, and one of them isthe actin-binding site. It can form a covalent bond to actin.
The second site is a catalyst that can split ATP. I'm goingto assume that you remember from basic biology that what happens,why is ATP there and why do we split ATP. Energy. ATP is the energycurrency of the cell. Mitochondria work really hard to makeATP. And then one of the reasons for making ATP, particularlyin the muscle cell, is that you can, then, power contraction.
That's what's going to happen. The myosin ATPase site is goingto split the ATP, get the energy out of the ATP and then use itin the process of powering muscle contraction.
Now, if you take the purified myosin with its tail section andits head sections, and you put a bunch of it together in a properPH and ionic strength, it will spontaneously arrange itself intothese thick filaments, which at one level it might look like that.
The idea is that you get a thick filament that has all the tailsections lined up parallel to one another, with the heads stickingout in 3 dimensions around the thick filament.
And the thick filaments and the thin filaments are arrangedin this fashion where they are sort of interdigitated with oneanother. A single thick filament has 6 thin filaments arrangedaround it in 3 dimensional space.
And we usually represent that on the black board or a pieceof paper as though there is just a thick filament with a thinone above and this thick one below it. You need to recognize thereare actually 6 arranged in a symmetrical fashion around eachindividual thick filament.
And that's the bundles, the bundles of thick and thin filamentswith a single thick filament with its head sticking out in 6 directionswith thin filaments in corresponding positions. And then anotherthick filament over here with 6 thin filaments around it, makingthese little hexagons.
That's like a cross-section through this structure right here.Take a look at this area where the thick and thin filaments areinterdigitated with one another. Make a cross section across thatand what you will see is this complex 3 dimensional array. Very,very regular arrangement of thick and thin filaments. And allof the thick filaments have the heads sticking out to the side.
And because those heads have an actin-binding site they canactually attach to the thin filaments and form what are called"cross bridges." So that if you take a muscle and youexercise it and deplete of it ATP, and then remove the muscleand section it, what you will see is that the thick filamentsare actually attached to the thin filaments.
A covalent bond formed between the actin-binding site on themyosin head and the filament. Another synonym for these myosinheads is "cross bridges" because they can form a crossbridge, a connection between a thick filament and thin filament.They are not always like that. They can form and break. And thatprocess we'll talk about on Friday.
However, before we start talking about the function of the muscle,we need to have a better understanding of 2 more intracellularorganelles.
What was the first intracellular organelle that I mentioned?
Myofibril.
The next of the -- there are 2 more intracellular organellesthat we need to know about in order to understand the functionof the cell.
This is another very sort of classical representation of the3 dimensional structure of a muscle cell. Again, it's just a partof it.
And one thing I want to emphasize to you is that I do not expectyou -- if I were to ask you the essay question on the functionof a muscle where I wanted you to describe the anatomy, internalstructure of the muscle cell, I would not expect you to reproducethis diagram.
They teach art over in Building 5. If you want to learn to bean artist, that's great. But we're going to do simple diagrams.So that's a thing may be useful for you to keep track of, whatdiagrams do you need to memorize how to do and what ones doyou not need to memorize how to do.
This one you don't need to memorize how to do.
This one, and some parts that I'll add to it on Friday, youdo need to memorize those diagrams. This is a 3 dimensional representationagain of a part of skeletal muscle cell. The dots recognized here-- here is a thick filament -- here are thin filaments arrangedaround it. That has been cut in a cross section so those dotsrepresent sections of this thin filaments.
This is bundle of thick and thin filaments. And this is a myofibril,and there is a myofibril, and there is a myofibril. So there isa whole bunch of these things.
The thick and thin filaments are really the functional partof the cell. They are part of the cell that we call the "contractalmachinery." They are the things that result in the forcebeing generated and the cell shortening. So another synonym thatI'll use for these is the "contractal machinery." That'sthe thick and thin filaments together.
So we can see that there are 2 more intracellular organelles.The first of these is called the "sarcoplasmic reticulum,"or the SR.
This is a membranous structure. It is composed of phospholipidbilayer. It is -- in an evolutionary sense, it's similar to endoplasmicreticulum that you learned in basic bio, although it doesn't havethe same kind of function. It's not rough ER.
It is -- in fact, it separates off within the interior of thecell in a separate fluid compartment contained within the SR.
And I have tried to represent that in a couple of ways. Theinterior of the SR -- the SR is the part I have colored red here.And the interior of the SR I have colored green in this area toshow that it's actually a separate fluid compartment within thecell.
And you can see in this diagram that it's wrapped around --the SR is wrapped around the myofibrils. Here in this very simplifieddiagram, you see the myofibril is this dark tubular structure,and the SR is wrapped around it.
Up here you can see the end, the SR actually isn't a solid sheetof material like that but it has these longitudinal tubular structures.But these little green dots that you can see here represent thecut off ends of this. So you can see the SR is wrapped aroundthe myofibril.
That's very important. It's wrapped around it because the signalto turn on the contractal machinery is going to be calcium comingout of the SR. And in order to activate all of the contractalmachinery simultaneously, you have to have the SR all wrappedaround it. You can't have just calcium coming from too far away.It has to diffuse. Calcium is going to come out of here and diffuseinto the interior of myofibril and activate that contractal machinery.It's very important, from a functional point of view, that theSR is wrapped around the myofibrils.
Now, the SR has these expanded areas. It has these little longitudinaltubular areas, but it has an expanded area that are called the"lateral sacs."
And these expansions of the SR, called the lateral sacs, arelocated in the immediate vicinity area of the intracellular organellethat we need to know about. And that's called a T-tubular, a transversetubule.
Abbreviated as T-tubule.
So the T-tubule, you'll notice they are labeled up here, rightthere, transverse tubule comes down like that. And notice on eitherside of it are these expansions, the lateral sacs of the SR. Hereis another T-tubule over here. Down here in this diagram theseT-tubules are these dark branches. They also branch and go aroundthe myofibrils.
And the T-tubules are little invaginations of the muscle cellmembrane. Invagination.
They are invaginations of the muscle cell membrane. That meansthe muscle membrane instead of just going across the surface ofthe cell, if you look up here for a second, here I am sort ofholding my hand against the sarcolemma. The sarcolemma is notflat like a ceiling, but it dives down into the interior of thecell forming, these little tubes that run through the interiorof the cell.
In fact, they come in and they branch around a myofibril andthey fuse together and then they branch around and go around anothermyofibril as well.
And there are literally thousands of them going along the lengthof the muscle cell itself, little invaginations of the musclecell membrane.
Now, if you have a little tubular invagination from the surfaceof the cell going down to the interior of the cell, what kindof fluid do you suppose is located in the middle of that tube?Is that fluid that's located in middle of that tube, is that goingto be high in sodium or high in potassium? In other words, isit going to be like the fluid outside the cell or is it goingto be like the fluid inside the cell?
STUDENT: Outside.
INSTRUCTOR: It's going to be the fluid outside the cell. It'shigh in sodium, low in potassium. It's extracellular fluid. Eventhough it's way in passing through the interior of the cell interms of the topology of the situation, it's really fluid fromoutside the cell.
And the phospholipid bilayer that makes up the T-tubule is continuouswith the muscle cell membrane. So what happens is when the actionpotential is traveling over the surface of the muscle cell, beingpropagated along by sodium channels opening and closing, thatsame thing happens and goes through the interior of the cell.
So the action potential does not stay on the surface of the cell;it actually dives through the interior of the cell traveling alongthe transverse tubules.
And it's very important that the lateral sacs are located immediatelyadjacent to the transverse tubules. Because remember I told you,calcium was going to come out of the SR. Well, it's this closeapproximation, there is almost contact between the T-tubule andlateral sac that allows the action potential to produce the releaseof calcium.
In fact, those structures were identified a very long time beforetheir significance was understood.
There was an old electron microspicus (spelling) who was studyingskeletal muscle, and he kept finding things that looked likethis -- a little round thing and a couple of oval things nextto it.
And he went to scientific meetings and he told people aboutthings called "triads." You know, because like mostscientists, he didn't want to say 3 little thingies. He wantedto have an official scientific sounding term, so he called them"triads." Which just means 3 little thingies.
So what are the 3 little thingies. The middle thing is what?A T-tubule. And what are the 2 oval shaped things on either sideof it? The lateral sacs of the SR. Here is a triad, right here.
Notice that these are not fused with one another. What is locatedin this space right here? Who knows?
STUDENT: Sodium.
INSTRUCTOR: That's a solution, that's extracellular fluid highin sodium. What's located in this space right here where my fingeris right now?
STUDENT: Muscle.
INSTRUCTOR: It's the interior of the muscle cell, the cytoplasmof the muscle cell. And what's located in this area right here?The fluid that's in the interior of the SR, which incidentallyat rest has a very high concentration of calcium.
Another ion that we're going to keep track of here. So thesarcplasmic reticulum in addition to being a phospholipid bilayerthat separates the fluid of the SR has 2 membrane-bound proteinsin it. It has calcium a pump, an energy consuming active transporterthat takes calcium away from the cytoplasm surrounding the contractalmachinery and sequesters it inside the SR.
And it has calcium channels. Normally closed calcium channelsso the calcium can't leak out. And what happens is that actionpotential that was spreading over the muscle cell membrane thatproduced by the sodium channels opening up, this action potentialright here, it goes down through the interior of the cell throughthe T-tubules.
And the lateral sacs of the SR are located in that general area.And that electrical signal causes the calcium channels to open.So calcium, which is at a high concentration, can diffuse outof the SR when the calcium channels in the SR open signalled bythe action potential and T-tubule, and that's causes contraction.And we'll talk about that process of contraction on Friday.
Vertebrate striated muscle, it is called "striated"because it has stripes on it. You may have been able to see thatin lab. They are fairly faint, but you can make them out withthe magnification we have available on the scopes in the lab.There is are not a substantial thing.
At a much higher magnification you would see something thatcorresponds to what you are seeing up here. There are alternatinglight and dark bands. Again, on the microscope deal that you hadin lab, you know, an individual muscle fiber might have lookedsomething like that, and the striations that you were lookingfor would have been something like that.
I mean, they would have been very, very, fine, very faint. Andwhat we're doing here is we're enlarging that one little tinyarea so that each one of those large lines is now a big thing.
So one of those dark lines correspondence to this area, magnifiedmaybe a thousands times more than it was in the microscopes thatyou were using in lab.
Those alternating light and dark bands result from the veryirregular arrangement of the thick and thin filaments in the muscleitself.
And the correspondence between that the alternating light anddark bands and the underlying molecular structure is what we'retrying to represent in this diagram.
The dark areas are called the A-bands. The "A" isshort for the word "Anisotropic" which refers to thefact that when polarized light is put -- it's used in one of themicroscope that you get a particular dark appearances. So thedark areas are the "A-bands." The light areas are the"I-bands."
In the middle of the I-band, that is the middle of the lightarea, you can see a very -- at high magnification, higher thanyou had in the lab, you can see a very faint line. That's calledthe "Z-line," and it is produced by a structural protein.
So I'm drawing a distinction here between a structural proteinand a contractal protein. The contractal are the thick and thinfilaments that are responsible for the contraction of the muscle.A structural protein is more like an internal skeleton, if youwill, a cytoskeleton.
It's something inside the cell that holds these things in thatregular arrangement. Remember that interdigitation with the thickfilaments having 6 thin filaments arranged around that in thisvery complex way. That's the result of these structural proteinsholding these thick and thin filaments in the proper location.
So the Z-line is a structural protein.
And the fourth area that can be identified sometimes is calledthe "H-zone." And that comes from the German word forbright, which is the word "hell" (spelling). So thereis a bright area that can be seen in the middle of the dark bandunder some circumstances.
And those circumstances are if the muscle was stretched, soyou cut the muscle out of the animal, you stretch it so that it'stowards a maximum kind of length that it might have been stretchedto in a living animal, and then you fix it and preserve it andsection it, then you'll see an H-zone.
If the muscle was stimulated so that it was contracted, andthen it was fixed and sectioned, you won't see the H-zone.
And the reason for that appearance and disappearance of theH-zone is depicted on the next page. But let's just take a lookat that page. So the A-band, dark area is where we find both thickand thin filaments overlapping one another, and that intuitivelymakes sense. There is a lot of protein so not as much light canget through.
The I-band is the opposite condition where there are only thinfilaments. So a maximum amount of light comes through becausethere is a smaller amount of protein. The Z -- line is a structuralprotein and the H-zone is where, under some circumstances, thethin filaments are not completely overlapping the thick filaments.
Right here in the middle is where the H-zone would be, wherethere is just a thick filament not both a thick and thin filament.
And that kind of observation really led to the understandingof how muscle contracts. Which is what's represented here.
On the upper part, we have a muscle in the stretched condition.In the lower part we have a muscle that has contracted some what.
And what you can see is the thin filaments slide past the thickfilaments. And so the H-zone disappears. That's how we can --why we get an H-zone when the thing is stretched, because thereis part of the thick filament that does not have thin filamentsoverlapping it.
And so some of the dimensions, that is some of the widths ofthese zones, which a microscopist with a high-powered microscopecould actually measure some of these dimensions change and someof them do not.
The width of the A-band, which is the length of thick filaments,does not change when a muscle contracts.
What does that tell us about the thick filament? It tells usthat the thick filament is not itself getting shorter.
Now, the significance of that observation is more obvious froma historical perspective. And that is when scientists first startedtrying to understand how muscle shortened, there were 2 competingmodels, 2 different hypotheses.
The one hypothesis was that what you had inside a muscle wasproteins that were in a way analogous to the proteins that arefound in a rubber band.
What happens with a rubber band is you stretch it, and you letgo of it. And when you stretch it there are proteins that arefolded, and when you let go of the end of the rubber band thething rekinks. And they are actually proteins molecules that arechanging their length.
And that was one of the hypotheses that some proteins insideof skeletal muscle, the proteins themselves were changing theirlength and producing this shortening.
Well, it turns out that hypothesis is wrong. And this kind ofobservation proves that it's wrong. The fact that the width ofthe A-band stays constant says that those proteins, at least,are not changing their length.
Now, the width of the I-band does change.
Does that mean that there is a protein change in length? No.What this shows us is that thin filaments are sliding past. Soyou should take a look at this diagram in your Illustrated Notesand make sure you understand which dimensions change length andwhy is it that some change length and some do not.
Of course, in subsequent years chemists and biologists havebecome a lot more knowledgeable about the actual process thattakes place and leads to the shortening of the muscle, and thisis what's called the "sliding filament mechanism." Itwas originally -- or hypothesis.
But what we now know is that this, in fact, is very, very welldocumented. Using many different, very, very, high tech, highspeed, assay systems, we know now quite a bit about how musclechanges length and what goes on.
And the term "sliding filament hypothesis" or "slidingfilament mechanism" is a little bit misleading, because sliding,you know, you think of sliding, you go slide down a slide at theplayground or go up to the mountains and you slide down a hillor toboggan or something is a fairly passive process.
In fact, what's going on in the case of muscle is very activeprocess. Remember that when I told you about the mechanisms orthe thick and thin filaments, I said there was a head part ofthe myosin molecule. And that head sticks out from the side ofthe thick filament. It has a place on it that can form a covalentbond to an individual actin molecule.
And so the actual sequence of events as the chemists understandit now involves -- they have sub-divide it, some of the 4 stepsthat I have here in your Illustrated Notes, because of carefulexamination of the details of the process. But this, I think,is a simplified version that's sophisticated enough that it'svery close to the details.
So if you understand this, and you get into a higher level biochemistryclass, you can appreciate some of subtilties of what is goingon here.
But basically what's happening is that the myosin head is forminga covalent bond to the actin. And the then the head itself isactually physically moving. It's kind of rotating through a smallangle. And because it has grabbed onto the thin filament, beforeit does that, it causes the thick and the thin filaments to moverelative to one another.
So if you look up here for a second, sort of act this out, ifthis is a thin filament and here is a thick with a myosin head,the sequence is that the myosin head attaches and then it rotatesand then it separates and then it returns, and then it attaches,and there are 4 steps in that cycle, in that process of events.And those 4 steps are repeated over and over again very, veryrapidly, so that as a consequences the filaments move past oneanother.
Now, if you look in the Illustrated Notes or you look at thediagram up here, you can see several different representationsof this sequence of events.
We can start in the upper left-hand corner where the myosinand the actin are separated from one another. And we can followthis around, if you look in the pictures here, they are separatedin the upper right-hand corner. What has happened is that themyosin head is now attached to the actin.
In the lower right-hand corner notice that angle between themyosin head and the thick filament has changed, that's the littlemovement step. In the lower left-hand corner the myosin head isstill tilted to left there, but it's separated from the actin.And then we get back up in the upper position, the myosin headis back in the vertical condition, but it's still separated fromthe actin.
Now, the names of those steps are indicated below here. Thefirst step is the binding step, that's where the myosin head attachesand forms a covalent bond to the actin using that actin bindingsite.
The second step is the movement step, where the filaments slidea very incredibly tiny molecular distance past one another asa result of the myosin head tilting.
The third step is the releasing step. When the myosin headbreaks its covalent bond to the actin.
And the forth step is the priming step. And that's called "priming"because what happens is that the myosin head gets energy fromATP. And it's kind of like cocking a trigger or priming a pump.You have to put we're into an old-fashioned pump to be able toget water out, well, that's preparing for the movement step.
That's one of the more surprising discoveries as a result ofthis research is that the time when the ATP splits is not thetime when the movement occurs.
Intuitively, people used to think, well, you know, ATP is beingsplit, energy is being used, and movement is occurring. You wouldexpect all those things to be happening simultaneously. But verycareful high-speed resolution shows that the splitting ATP occursin a different step in this process, and that's the priming step.
Another thing that's interesting is you'll see that ADP andinorganic phosphate which is the piece of I, the ADP and inorganicphosphate split off during the movement step, and that duringthe releasing step is when ATP attaches. And it turns out thatthat is a necessary part of this releasing. There has to be ATPavailable in the cell for the covalent bond between the actinand myosin to break.
You may be aware of the fact that when an animal dies, its musclesare very lacid, it's limp, its body is limp. After it sits aroundfor a period of time it becomes very stiff.
That technically the called rigormortis, that's Latin for thestiffness of death. And rigamortis results from the fact the cells-- the ATP supply in the cells is depleted. And there is no longerany ATP to cause a splitting of this to cause this releasing stepto occur.
So the muscle is stiff because the actin and the myosin is arebound to one another, and therefore the muscle cannot change length,and the muscle in the body of the animal the stiff.
Now, for those of you that are into chemistry in parallel withsequence of pictures are some chemical reactions. I'm not goingto hold you responsible for the details of those chemical reactions.But, you know, for those of you who are -- that may be helpfulin terms of understanding.
The idea here is -- notice upper left-hand corner the "M"has a little asterisk next to it. That means it's high energymyosin. It reflects the fact that it has received that energyfrom the ATP and is prepared to use it. And where you have "A"plus "M" that means that actin and myosin are separatedfrom one another. Whereas over in the upper right-hand corneryou have "A" dot "M," and that does representsthe covalent bond between the two.
So you can see binding represented chemically as we go from"S" plus "M" to "A" dot "M"you can see the use of energy by the myosin when we go from "M"star in the upper right-hand corner to just plain "M"in the lower right-hand corner.
Any questions about that? That is called a cross-bridge cycle.Remember the cross-bridge is the attachment of the myosin to theactin by means of the myosin head. So this is a cycle that isrepeated over and over again very rapidly hundreds of times inorder to cause a muscle cell or a muscle fiber to shorten.
STUDENT: So, like, at resting position, the myosin head wouldbe bound to the actin?
INSTRUCTOR: No. In a dead animal myosin would be bound to theactin. In rigamortis that actin and myosin would be bound to oneanother. In the resting state, when the muscle cell is not active,the myosin is in the primed condition, it's the high energy myosin,but it is not bound to the actin. It's not attached to the actin.
That's the resting condition.
You pull on the end of that muscle cell it just lengthens dueto stretching.
The resting state, there is no attachment between the actinand the myosin.
Now, what I want to do is I want to put this -- a whole processtogether into a single diagram so that you can see how the wholething works. So I'm going to reproduce part of the diagram youhave already seen, and not spend a lot of time talking about it.
Here is the axon terminal. Here is the sort of indented areain the muscle cell. Those of you who are getting ready to copythis diagram are doing the right thing.
This is a diagram that appears on midterm exams every once ina while. There is the sarcolemma. There is the T-tubule.
Now, remember that the sarcoplasmic reticulum is wrapped aroundthe myofibrils which are composed of thick and thin filaments.So trying to diagram that is more complicated. What I'm goingto do is separate those 2 parts of the machinery from one another.
So here is a couple of T-tubules, and here we are going to havethe lateral sacs and the SR -- remember there is a high calciumconcentration in here. We are going to add in our membrane-boundproteins. We have got synaptic vesicles with acetylcholine. Wehave the ACH receptor. We have an integral protein, functionallyin the diagram as being separate, but really integral.
What's this? Sodium channel. And that's one that's normallyclosed, or voltage -- this is actually gated by the -- it's openedwhen ACH binds to the receptor.
We also have another membrane-bound protein in neuro muscularjunction. What's this?
ACHase, the enzyme that catalyzes the breakdown of acetylcholine.We have some other -- over here we have, what is this symbol forhere? The standard physiologist's diagram of a pump is a little-- like a wheel. You think about a little powered motor with anarrow indicating the direction in which calcium is going to bepumped.
And another channel, in this case a calcium channel, also normallyclosed.
And then we are going to add into this picture, something thatwould be sort of -- that this would be wrapped around, and that'sgoing to be our representation of the contractal machinery. Sohere is the standard representation of the contractal machinery,the thin filament and the thick filament.
All right. So I'm not labelling everything in this diagram thatI have already labeled for you in the preceding diagram of theneuro muscular junction.
But I'm going to add in here, here's the T-tubule. Here's thelateral sac of the sarcoplasmic reticulum. This is Z-line, thestructural protein, the thick filaments, and thin filaments, thecalcium channels. And this is interior of the SR.
Now, what we have here, when we go from one Z-line to the nextZ-line, we have the T-tubules and the SR, is what's called a sarcomere.This set of structures is a sarcomere. Included in that are theall the bits and pieces that we need to understand in order tounderstand how a muscle cell works.
A single muscle fiber will have hundreds of sarcomeres alongits length. They will all be exact replicas of this. And whathappens in one is happening simultaneously in all of them.
So a tiny little bit of shortening on the order of a molecularkind of distance, repeated by thousands of times, by thousandsof sarcomeres in series with one another along the length of themuscle cell is what results in a large measurable shortening ofthe muscle cell.
And the sequence of events are the following: The action potentialcomes down the axon, it causes exocytosis from the synaptic vesiclesthat release the acetylcholine into the synaptic cleft. The acetylcholineis going to diffuse across the cleft and combine with the acetylcholinereceptor.
The receptor is going to cause a sodium channel to open so thatsodium flows into the cell, and that causes the membrane to depolarizeand generate and action potential. The action potential is goingto propagate over the sarcolemma, the muscle cell membrane. It'sgoing to go through the interior of the cell through the T-tubules.
As it goes past the lateral sacs of the sarcoplasmic reticulum,it's going to cause calcium channels to open. The calcium willdiffuse out of the SR into the sarcoplasm, that is the cytoplasmof the muscle cell. And the calcium, by mechanism I'm not goingto into the details of, allows the first step in a cross-bridgecycle.
It actually allows the binding step to occur. There are someother proteins that are on the thin filaments which prevent thatbinding step from occurring in the resting condition. For thoseof you that have had some more advanced physiology, I'm talkingabout troponin and tropomyosin at this point, but that's beyondthe scope of the course.
So when the calcium is released, it allows the initiation ofthe cross-bridge cycle. And the cross-bridge cycle occurs hundredsof times, and the thick and thin filaments slide past one another,and that constitutes a twitch, a single shortening of the contractalmachinery in a muscle cell.
Finally, the whole signal gets turned off, remember the acetylcholineesterase is going to break the acetylcholine down into cholineand acetate. And that causes the membrane to repolarize. And thenthe cell has to have some force exerted on the end so that itstretches back out again.
Muscles can only actively shorten. For a muscle cell to lengthensomebody has to pull on the end of it. And muscles are arrangedin the body of a vertebrate animal in antagonistic pairs. So Ihave my biceps which contracts and causes this to occur. And Ihave my triceps which operates on the other side of the jointof my elbow and pulls the muscle back.
So this muscle shortens, the triceps shortens and pulls thatway, the biceps shortens and pulls that way. These 2 muscles constitutean antagonistic pair and cause joints to operate in opposite direction.
STUDENT: So the lateral sacs in the neuro junction cell is thatwhole area?
INSTRUCTOR: Well, no, I'm not trying -- this same thing couldbe repeated over and over again along the entire length of themuscle. So the SR, itself, will be repeated as well as the thickand thin filaments and Z-lines.
STUDENT: Will the pumps also occur in the sarcomere or do theyjust appear next to the neuro muscular junction?
INSTRUCTOR: Which pumps?
STUDENT: The pumps --
INSTRUCTOR: So far have we have only looked at one pump, whichis in the membrane of the SR. This same thing, there would beanother here, and another here, and all the way up to somewherein West Covina someplace.
So the same thing is happening simultaneously. This wave depolarizationis going to spread over the whole entire muscle cell, all thealong the whole entire length.
And it's going to go zinging through the interior of the celldown these transverse T-tubules, all over the whole entire lengthof the cell, causing calciums to be released through all the subsectionsof SR along the entire length of the cell, and all the sarcomeresare going to be active. So the whole entire thing happens simultaneously.
Any other questions about that? All right.
So as an example of the kind of essay question that you shouldbe prepared to answer on your midterm, which is in, like, a weekand a half from now, would be describe the sequence -- diagrama neuro muscular junction and the contractal machinery insidea muscle cell. That means reproduce this diagram and label it.
And then the other part would be describe the sequence of eventsthat occurs between the arrival of an action potential at theaxon terminal and the completion of contraction by the musclecell.
And then you would just say everything that I just said about-- et cetera, et cetera. And that would be a 30-point essay question.
STUDENT: How do the T-tubules cause the calcium to be released?
INSTRUCTOR: That's a really interesting question and unfortunatelyit's beyond the scope of the course right now. But there are these-- the calcium channels have, like, a little plug in them. Andthe plug is pulled electrically. But there are also located herein the immediate proximity to the T-tubules. The plug is kindof pulled out, and then some calcium comes out.
And then that calcium feeds back on the channel and causes allthe channels to open up. So the calcium which is going throughthe channel also has a receptor site on the channel. It's reallya marvelously complicated system. And it's more complicated thanthat.
But it is the electrical signal in the T-tubule is what triggersthe opening of the calcium channels.
STUDENT: And the fibers that are thick and thin, they are locatedall throughout, not just in the --
INSTRUCTOR: You remember the diagram that showed the SR waswrapped around the tubules, the myofibril. The myofibril is bundlesof thick and thin filaments. If we look back at this diagram here.
Here is that same picture.
Here is the Z-line. Here is the thin filaments. There are thethick filaments. That's the same diagram I have drown there. Andthen these are the lateral sacs of the SR. Remember these littlethings around here? Like that, the SR is wrapped all the way aroundthat. And the T-tubule is coming down.
Remember this diagram down here shows that the T-tubule actuallyis branching around that as well.
When I said I was drawing this down here, because it would betoo complicated to try and draw it behind this, but it actuallyis behind this.
Any other questions?
STUDENT: The action potential goes through there and that causesthe sacs to --
INSTRUCTOR: Calcium channels.
STUDENT: The --
INSTRUCTOR: Remember, the calcium is what allows that firststep of the cross-bridge cycle to occur. So that the thin andthick filaments slide past one another. You have to get the calciumback into the SR before the muscle can relax. As long as thereis calcium around the thick and thin filaments, they are goingto stay in a contracted state.
They are going to slide past one another and then they are goingstay there, locked up, in that contracted state.
Now, we know that our muscle doesn't normally function by twitching;otherwise I'd be jerking around up here. Muscle normally functionsin what is called a titanic contraction, it maintains a constantforce. It does that by being repeatedly stimulated by the nervoussystem so that there is always some calcium in there, and themuscles, in fact, are not going through a twitching process, theyare staying in that contracted state.
That's sort of beyond the scope of the course.
But the point is the calcium pump, in order for that muscleto relax so that it can be lengthened, the calcium pump has toput the calcium back into the SR and get it away from the thinfilaments, so that binding stops.
STUDENT: The lateral sacs and the SR, it's right on top of themyofibrils? It's right -- it's like here is the myofilaments andhere is my hand representing, sitting on top of each other.
INSTRUCTOR: It's wrapped around it. Remember this diagram downhere. There is the myofibril. And the green shows the edge ofit is wrapped all the way around it, so that when the calciumis released from the SR, it's permeating into the interior ofthat myofibril from all 360 degrees, wrapped all the way aroundit.
The calcium pump is always active. Anytime it can get calcium,it's going to bump it. So it's like, whenever there is substratepresent then it's active.
So as soon as the calcium is released from the channels, thepump starts putting it back again as fast as it can. Calcium issitting there, and some calcium ions are going loop to loop likelittle mad mother's, going around and around, one guy gets out,and the pump grabs him and sticks him back inside again.
So he back gets out, and he gets stuck back inside again.
There is a war going on between the calcium that's trying todiffuse out down the concentration gradient and the calcium pumpthat says, "No, you are in here man. Get away from that thinfilament."
STUDENT: Does the normally closed sodium channel that's openafter the ACH opens, does that cause the sodium to leak from thesynaptic cleft or from the sarcoplasmic reticulum?
INSTRUCTOR: There is calcium in the sarcoplasmic reticulum.There is sodium outside the cell.
So the sodium and I have only drawn -- now, remember that anaction potential and a nerve cell is regenerated along the lengthof a nerve cell. When I say it's propagated -- when I say theaction potential is along the muscle cell, the same thing is happening.
So there are sodium channels up here, and along here, and alonghere. Okay. Sodium channels all along that whole entire length.And when this section of membrane depolarizes so that the insidemembrane is at rest, this stuff is negative on the inside andpositive on the outside.
But when these sodium channels up, then this reverses the polarity,and by a mechanism I haven't described yet, but it involves thelocal flow of ions. The membrane over here becomes depolarizedand these sodium channels -- remember talking sodium channelssitting there in the membrane with a charge across them and it'sswitches? Well, that causes them to open.
And so then we get an action potential generated in this area,this triggers an action potential to be generated in this area,which also travels down the T-tubules.
So sodium channels, along the whole entire length of the sarcolemma,the muscle cell membranes, are being triggered to open and generatean action potential. The action potential, then, causes the calciumchannels in the SR to open the calcium comes out.
STUDENT: The T -- tubules have sodium calcium in them?
INSTRUCTOR: Right. Remember I made that point about this fluidout here that's high in sodium, the extra cellular fluid, thatstuff is also in the T-tubules.
STUDENT: It's not part of the SR?
INSTRUCTOR: No.
Remember the triads had a T-tubule, 2 T-tubules and an SR nextto them. We make a little cross-section across here, and we getsomething that looks like that. That's separate. So we have highsodium in the middle of a T-tubule; high calcium in the SR.