BIOL 201: lecture 22
BIOL 201: lecture 22--> march 1st 2010.
Different Types of Actin Networks • How do cells make different networks in different places? • How do cells decide which network to put where?
There are several diff types of actin network Leading edge, branched actin networl composed mostly of Arp2/3, filapodia that are projections that cells can shoot out, out f the cells we have these cell fibers or actin bundles.
Actin Networks: Listeria
Actin networks formed by Arp2/3 complex ( shown here in light blue)--> branched daughter filament that comes out at a 70 deg angle.
Capping protein ( shown here in green)--> caps the end of a growing actin filament and prevents for a subunit addition--> thatʼs important to keep actin polymerization at the filaments. Cofilin is an actin severin enzyme--> itʼs cutting these actin filaments while accelerating the disassembly so that you have enough power to keep actin polymerization forward.
Actin Networks: Leading Edge
Apr2/3 complex is forming branched actin networks. Capping proteins are capping the filaments that you donʼt want to polymerize anymore. Profilin helps catalyze the exchange of ADP out of actin and replacing it with ATP--> important for recycling your actin away from the - ends back to the leading edge
Grow the Filaments: Formins
Formins are actin polymerazes--> they polymerize enzymes... the FH2 domain is a chimeric molecule that climbs around the end of a growing actin filament and changes the rate at which it elongates and specifically controls the actin elongation. --> Also there are actin cross-linkers. Theyʼre proteins with bundle actin filaments in specific ways. ( check previous lecture)
Cross-link the Filaments
The amoeba is extending its leading edge, itʼs extending itʼs Arp2/3 complex i the ddirection of the cyclic AMP needle... itʼs then forming stress fibers. Itʼs using that to drag itself towards the end of its food.
Chemotaxis
Chemotaxis
Extension of the lamellipodium ( also called the leading edge). It reaches out to form a new adhesion, the cell made a new firm contact with its extracellular environment...etc...
Rho Family GTPases
Signalling GTPases--> Rho family. Theyʼre the master regulators of cell cellular actin morphology. Theyʼre the molecules that are transducing signals from the extracellular environment and that regulate all the other actin regulators. Dominant active is a molecule thatʼs always in the on state. microinjection of 3 diff molecules: Rho, Rac and Cdc42.
They caused 3 very dramatic and diff underlying effects on the cytoskeleton. This is very conclusive demonstration. So these Rho family of GTPases are master regulators of actin cytoskeletal organization. In the case of Rho the signal is: “form stress fibers” --> General phenomena of GTPase phase signalling.
GTPase Paradigm
You have a molecule that can have 2 nucleotides in its nucleotide binding pocket--> it can have GDP or GTP. GDP form is the inactive form.--> when molecule is in this state the signalling is turned off. When the molecule is pushed into the GTP state--> active form of the molecule and the molecule is turned on. Rho in the GTP state is therefore telling the cell to form stress fibers. So dominant active forms are forms of the protein that are constantly in the GTP state. These proteins cannot hydrolize the GTP. In the absence of any signalling Rho is in the GDP state. It has GDI (guanasine diphosphate inhibitor) which holds Rho in the GDP state--> itʼs keeping it under lock and key. Then an extracellular signal comes in to some receptor thatʼs on the plasma membrane and in this case it would be that cAMP. the cell now knows that it needs to start turning on because the binding of the extracellular signal to the receptor activates a molecule called GTP exchange factor ( GEF). this factor finds Rho GDP, takes the GDP out and sticks the GTP in. Rho is no on and stimutates the formation of stress fibers. To turn all of this off to stop forming RhoGTP thereʼs a protein called GTPase activating protein (GAP) that catalyse the hydrolysis of the GTP and convert the RhoGTP back into RhoGDP. They turn the signal off.
Somehow this RhoGTP signal has to be able to do something... The on state of the signalling molecule has to be able to reach out and tell something to start working... this is hat the effector proteins are here for. Theyʼre downstream of RhoGTP.
Three pathways
The 3 diff GTPases give 3 diff actin output. Effector proteins: WASP, WAVE, Formin.--> they turn a specific GTPase into a specific type of actin cytoskeleton polymerization.
Cdc42 activates Arp2/3
One of the activators that translate the Cdc 42 signal into fillipodia formation is a protein called WASP.WASP has an RBD(rho binding domain). But it doesnʼt actuall bind Rho, it
binds Rho family proteins and rho binding domains are a generic term for structural domain that binds to a Rho family GTPase. WASP is in a folded state, itʼs closed upon itself and the real critical domain ( blue and orange) is going to turn on Arp2/3. But theyʼre hiden because the protein is folded up on itself and in a closed configuration. The Rho binding domain has the ability to bind to Cdc 42 GTP ( and only that one )... so thereʼs an activation: The binding domain bind sto the Cdc 42 GTP--> that causes the molecule to open up. now the Arp2/3 binding domains are now free and can activate the Arp2/3 complex shown here by the direct contact of the blue domain with the Arp2/3 domain and the interaction of the orange domain with an actin monomer.
Rho activates Formins
Formins also have an RBD.... its mainly the same thing as above. Inactive form of the GTPase is directly interacting with the activator molecules. The speed of the response has to be quite fast. RowGTP binds formins and formins go.
Thereʼs a Cdc42 activation at the front. Rac activation also--> these 2 lead to fillipodia and lamellipodia formation. Rho is now active at the back--> leading to the formation of stress fibers. ( also called contractile bundles.) But all this is only downstream. At the very front of the sensing of the chemotactic gradient, thatʼs the region where cdc42 is active.
In absence of any of one of the regulators the whole motile process shuts down. you need all 3.
Donʼt worry about memorizing all the names here.
There are some molecules like the surface receptors that never themselves become polarize. Theyʼre always continuously around the cell at diff areas of the membrane... Once the cell starts sensing a change in the gradient... or in the direction it wants to go you get a very rapid polarisation of the cell where some lipids and signalling molecules end up being polarised.( kinase, phosphatase, actin...)--> read in the textbook. uniform distribution of the receptors gives rise to a very rapid polymerization of a large number of molecules. These gradients are often very sutle ( 2%-->5% variation between the 2 ends of the grandient.) The system therefore has to be ery finetuned, very sensitive to very small diff in extra-cellular signal. That implies that there are positive and negative feedback loops scuch that very small diff in signal get amplified into large changes in the distribution of intracellular proteins.
Stress Fibers and Contraction
When a cell wants to detach from a surface it has to break chemical bonds. This is a process that requires force, energy. A bundle of actin filaments gets shorter, they slide relative from one another. The bundle gets thicker and denser in the process.-> thatʼs what happens when you flex a muscle. The blue is myosin.
Myosin: a Molecular Motor
Motor protein: protein that is capable of producing force. This is a bundle of myosin.
Myosin: a Molecular Motor
Crystal structure of myosin. It has a nucleotide binding site ( meaning that it binds ATP) --> it binds ATPase. Source of the energy is ATP, and the molecule is able to couple the hydrolysis of ATP into a large scale conformational change that causes the motor to change shape in such a way that you get contraction.
Myosin Moves Actin Filaments
Put the head of hte myosin on a glass slide. Filament 1, 2, and 3 move across the surface. So these proteins have the ability to translocate actin filaments. The head has ATP in it, it hydrolizes ATP and converts the hydrolysis in to a power stroke. it pushed the actin filament along when it does that, and it translocates actin filaments. There are diff classes of myosin.. the most critical is the class 2 myosins.
Types of Myosin
Class II myosins are dimeric molecules that form bundles of myosin motors. And theyʼre able to pull out the actin bundles in which theyʼre embeded. When a cell is migrating, those myosins that are in the stress fibers reach out at the actin, which is connecting down into the rear adhesion. So the myosins hydrolyze ATP and they tear up the back food.
Contraction and Muscle
The myosins grab on to the actin filaments and drive them. Contracion--> the structure gets shorter. Contraction and Muscle
• Structure of Muscle Fibers • How Myosin performs Work • The Sliding Filament Model
Different Types of Actin Networks • How do cells make different networks in different places? • How do cells decide which network to put where?
There are several diff types of actin network Leading edge, branched actin networl composed mostly of Arp2/3, filapodia that are projections that cells can shoot out, out f the cells we have these cell fibers or actin bundles.
Actin Networks: Listeria
Actin networks formed by Arp2/3 complex ( shown here in light blue)--> branched daughter filament that comes out at a 70 deg angle.
Capping protein ( shown here in green)--> caps the end of a growing actin filament and prevents for a subunit addition--> thatʼs important to keep actin polymerization at the filaments. Cofilin is an actin severin enzyme--> itʼs cutting these actin filaments while accelerating the disassembly so that you have enough power to keep actin polymerization forward.
Actin Networks: Leading Edge
Apr2/3 complex is forming branched actin networks. Capping proteins are capping the filaments that you donʼt want to polymerize anymore. Profilin helps catalyze the exchange of ADP out of actin and replacing it with ATP--> important for recycling your actin away from the - ends back to the leading edge
Grow the Filaments: Formins
Formins are actin polymerazes--> they polymerize enzymes... the FH2 domain is a chimeric molecule that climbs around the end of a growing actin filament and changes the rate at which it elongates and specifically controls the actin elongation. --> Also there are actin cross-linkers. Theyʼre proteins with bundle actin filaments in specific ways. ( check previous lecture)
Cross-link the Filaments
The amoeba is extending its leading edge, itʼs extending itʼs Arp2/3 complex i the ddirection of the cyclic AMP needle... itʼs then forming stress fibers. Itʼs using that to drag itself towards the end of its food.
Chemotaxis
Chemotaxis
Extension of the lamellipodium ( also called the leading edge). It reaches out to form a new adhesion, the cell made a new firm contact with its extracellular environment...etc...
Rho Family GTPases
Signalling GTPases--> Rho family. Theyʼre the master regulators of cell cellular actin morphology. Theyʼre the molecules that are transducing signals from the extracellular environment and that regulate all the other actin regulators. Dominant active is a molecule thatʼs always in the on state. microinjection of 3 diff molecules: Rho, Rac and Cdc42.
They caused 3 very dramatic and diff underlying effects on the cytoskeleton. This is very conclusive demonstration. So these Rho family of GTPases are master regulators of actin cytoskeletal organization. In the case of Rho the signal is: “form stress fibers” --> General phenomena of GTPase phase signalling.
GTPase Paradigm
You have a molecule that can have 2 nucleotides in its nucleotide binding pocket--> it can have GDP or GTP. GDP form is the inactive form.--> when molecule is in this state the signalling is turned off. When the molecule is pushed into the GTP state--> active form of the molecule and the molecule is turned on. Rho in the GTP state is therefore telling the cell to form stress fibers. So dominant active forms are forms of the protein that are constantly in the GTP state. These proteins cannot hydrolize the GTP. In the absence of any signalling Rho is in the GDP state. It has GDI (guanasine diphosphate inhibitor) which holds Rho in the GDP state--> itʼs keeping it under lock and key. Then an extracellular signal comes in to some receptor thatʼs on the plasma membrane and in this case it would be that cAMP. the cell now knows that it needs to start turning on because the binding of the extracellular signal to the receptor activates a molecule called GTP exchange factor ( GEF). this factor finds Rho GDP, takes the GDP out and sticks the GTP in. Rho is no on and stimutates the formation of stress fibers. To turn all of this off to stop forming RhoGTP thereʼs a protein called GTPase activating protein (GAP) that catalyse the hydrolysis of the GTP and convert the RhoGTP back into RhoGDP. They turn the signal off.
Somehow this RhoGTP signal has to be able to do something... The on state of the signalling molecule has to be able to reach out and tell something to start working... this is hat the effector proteins are here for. Theyʼre downstream of RhoGTP.
Three pathways
The 3 diff GTPases give 3 diff actin output. Effector proteins: WASP, WAVE, Formin.--> they turn a specific GTPase into a specific type of actin cytoskeleton polymerization.
Cdc42 activates Arp2/3
One of the activators that translate the Cdc 42 signal into fillipodia formation is a protein called WASP.WASP has an RBD(rho binding domain). But it doesnʼt actuall bind Rho, it
binds Rho family proteins and rho binding domains are a generic term for structural domain that binds to a Rho family GTPase. WASP is in a folded state, itʼs closed upon itself and the real critical domain ( blue and orange) is going to turn on Arp2/3. But theyʼre hiden because the protein is folded up on itself and in a closed configuration. The Rho binding domain has the ability to bind to Cdc 42 GTP ( and only that one )... so thereʼs an activation: The binding domain bind sto the Cdc 42 GTP--> that causes the molecule to open up. now the Arp2/3 binding domains are now free and can activate the Arp2/3 complex shown here by the direct contact of the blue domain with the Arp2/3 domain and the interaction of the orange domain with an actin monomer.
Rho activates Formins
Formins also have an RBD.... its mainly the same thing as above. Inactive form of the GTPase is directly interacting with the activator molecules. The speed of the response has to be quite fast. RowGTP binds formins and formins go.
Thereʼs a Cdc42 activation at the front. Rac activation also--> these 2 lead to fillipodia and lamellipodia formation. Rho is now active at the back--> leading to the formation of stress fibers. ( also called contractile bundles.) But all this is only downstream. At the very front of the sensing of the chemotactic gradient, thatʼs the region where cdc42 is active.
In absence of any of one of the regulators the whole motile process shuts down. you need all 3.
Donʼt worry about memorizing all the names here.
There are some molecules like the surface receptors that never themselves become polarize. Theyʼre always continuously around the cell at diff areas of the membrane... Once the cell starts sensing a change in the gradient... or in the direction it wants to go you get a very rapid polarisation of the cell where some lipids and signalling molecules end up being polarised.( kinase, phosphatase, actin...)--> read in the textbook. uniform distribution of the receptors gives rise to a very rapid polymerization of a large number of molecules. These gradients are often very sutle ( 2%-->5% variation between the 2 ends of the grandient.) The system therefore has to be ery finetuned, very sensitive to very small diff in extra-cellular signal. That implies that there are positive and negative feedback loops scuch that very small diff in signal get amplified into large changes in the distribution of intracellular proteins.
Stress Fibers and Contraction
When a cell wants to detach from a surface it has to break chemical bonds. This is a process that requires force, energy. A bundle of actin filaments gets shorter, they slide relative from one another. The bundle gets thicker and denser in the process.-> thatʼs what happens when you flex a muscle. The blue is myosin.
Myosin: a Molecular Motor
Motor protein: protein that is capable of producing force. This is a bundle of myosin.
Myosin: a Molecular Motor
Crystal structure of myosin. It has a nucleotide binding site ( meaning that it binds ATP) --> it binds ATPase. Source of the energy is ATP, and the molecule is able to couple the hydrolysis of ATP into a large scale conformational change that causes the motor to change shape in such a way that you get contraction.
Myosin Moves Actin Filaments
Put the head of hte myosin on a glass slide. Filament 1, 2, and 3 move across the surface. So these proteins have the ability to translocate actin filaments. The head has ATP in it, it hydrolizes ATP and converts the hydrolysis in to a power stroke. it pushed the actin filament along when it does that, and it translocates actin filaments. There are diff classes of myosin.. the most critical is the class 2 myosins.
Types of Myosin
Class II myosins are dimeric molecules that form bundles of myosin motors. And theyʼre able to pull out the actin bundles in which theyʼre embeded. When a cell is migrating, those myosins that are in the stress fibers reach out at the actin, which is connecting down into the rear adhesion. So the myosins hydrolyze ATP and they tear up the back food.
Contraction and Muscle
The myosins grab on to the actin filaments and drive them. Contracion--> the structure gets shorter. Contraction and Muscle
• Structure of Muscle Fibers • How Myosin performs Work • The Sliding Filament Model
