q3 cell

What are some important functions of the cytoskeleton within the cell?

The cytoskeleton maintains cell shape, facilitates cell adhesion, and is involved in the intracellular transport of proteins and organelles. It also helps with cell movement and is responsible for organizing and segregating chromosomes in cell division.

What are some important functions of intermediate filaments within a cell? What are some structural characteristics of intermediate filaments? For this question, think about how structure and function are connected.

Intermediate filaments are rope-like fibers that are composed of elongated proteins that maximize lateral interactions which makes intermediate filaments very strong and flexible. Intermediate filaments are responsible for providing mechanical strength to cells which is important for cell shape and adhesion. They are also involved with cell division because intermediate filaments called nuclear lamins are disassembled in late prophase and reassembled in telophase.

Describe the subunits and protofilaments in intermediate filaments. How are they used to assemble intermediate filaments? How many protofilaments are in an intermediate filament?

Different types of intermediate filaments are made from subunits that share a common structure. Subunits for intermediate filaments are observed as two elongated proteins (two monomers) coiled around each other to form a homodimer. Protofilaments are formed when two homodimers form a staggered tetramer with non-polarized ends (both ends of staggered tetramer have amino groups from N-termini) because the homodimers are antiparallel. Eight staggered tetramers/protofilaments pack together to form a small section of the intermediate filament. Other bundles of protofilaments can be attached to make the intermediate filament longer using the non-polarized ends.

what are nuclear lamins? What is the function of nuclear lamins within a cell? What happens to nuclear lamins during mitosis?

Nuclear lamins are the intermediate filaments that are found under the inner nuclear membrane to form the nuclear lamina. During late prophase in mitosis nuclear lamins in the nuclear lamina are phosphorylated by kinases to disassemble the nuclear envelope. After telophase in mitosis, the lamins are dephosphorylated by phosphatases to reform the nuclear lamina and nuclear envelope.

What are some important functions of microtubules within a cell? What are some structural characteristics of microtubules? For this question, think about how structure and function are connected

Microtubules are hollow cylinders that are stiffer (not as flexible) and more unstable. Part of the reason for the instability is that microtubules are made of globular proteins that have reduced lateral interactions. Microtubules are involved with intracellular transport because proteins and organelles can be localized to specific locations with stiff structures. Microtubules are involved in cell division because they are used to make the mitotic spindle that binds to chromosomes so that they can be segregated at anaphase. Microtubules also form cilia and flagella that are important for cell movement in a liquid environment.

Does a microtubule have a similar structure to an intermediate filament or is it different?

They are different structures. Intermediate filaments are ropes with non-polar ends while microtubules are hollow cylinders and have polar ends. The polar ends make microtubules very dynamic, allowing them to grow and shrink, which is critical when they assemble to bind to chromosomes during late prophase and metaphase then disassemble to segregate chromosomes in anaphase.

Why are intermediate filaments stable? Why are microtubules and actin filaments dynamic? Provide at least two reasons for this difference.

Intermediate filaments are stable because they have nonpolar ends making them more resistant to disassembly and assembly. Microtubules and actin filaments are dynamic because they have polar ends allowing growth on the plus ends of each structure. Additionally, intermediate filaments are composed of elongated proteins that use a high number of lateral interactions to form a stable structure. Microtubules are composed of globular proteins which are easier to disassemble.

What is tubulin? Describe the difference between β and α tubulin.

Tubulin is a heterodimer protein (quaternary structure) consisting of two subunits. Both beta and alpha subunits bind to GTP but only the beta tubulin can hydrolyze GTP to GDP.

Describe the subunits and protofilaments in microtubules. How are they used to assemble microtubules? How many protofilaments are in a microtubule? What is the minus end of a protofilament? What is the plus end of a protofilament?

The subunits for microtubules are two globular proteins (beta and alpha tubulin) that combine to form a heterodimer. One protofilament is created when heterodimers are stacked on top of each in the same orientation (beta subunit on top, alpha subunit on bottom). The protofilament has polarized ends because the beta subunit is observed at one end and the alpha subunit is observed at the other end. Thirteen protofilaments are arranged in a circle to create the hollow cylinder structure of a microtubule. All thirteen protofilaments have a beta subunit at one end that forms the plus end while at the other end all thirteen protofilaments have an alpha subunit that forms the minus end. Plus ends of microtubules always grow faster than the minus end of microtubules.

Describe the significance of GTP hydrolysis in a microtubule.

When the tubulin dimer is not incorporated into a microtubule it is bound to GTP but the rate of GTP hydrolysis in the beta subunit is slow. When a tubulin dimer becomes incorporated into a growing microtubule, the rate of hydrolysis in the beta subunit increases. GTP hydrolysis in the beta subunit is important for growth and disassembly of microtubules because it regulates the shape of heterodimers in a microtubule which influences lateral interactions with other heterodimers in the microtubule. GTP-bound heterodimer = lots of lateral interactions with surrounding heterodimers (stable); GDP-bound heterodimer = decrease in lateral interactions with surrounding heterodimers (less stable)

What is a GTP cap? Describe how a GTP cap influences polymerization and depolymerization of microtubules.

A GTP cap is an aggregate of GTP-bound heterodimers at the plus end of a microtubule. When a microtubule has a GTP cap it is growing because the rate of adding tubulin heterodimers to the plus end is faster than the rate of hydrolysis by the

What is a GTP cap? Describe how a GTP cap influences polymerization and depolymerization of microtubules?

A GTP cap is an aggregate of GTP-bound heterodimers at the plus end of a microtubule. When a microtubule has a GTP cap it is growing because the rate of adding tubulin heterodimers to the plus end is faster than the rate of hydrolysis by the beta subunit. If the rate of adding tubulin heterodimers to the plus end of a microtubule decreases there will be an aggregate of GDP-bound heterodimers which will encourage microtubule shrinkage.

What is dynamic instability? What is the association between dynamic instability and the GTP cap?

Dynamic instability is the process of a microtubule rapidly switching between growing and shrinking. When a GTP cap is present at the plus end of a microtubule, polymerization (growth) occurs. When a GTP cap is not present at the plus end of a microtubule, depolymerization (shrinkage/catastrophe) occurs.

What needs to be in a test tube to promote microtubule polymerization? There were two important components in this reaction that were discussed in class.

Growing microtubules in a test tube is described as in-vitro polymerization because it represents spontaneous generation of microtubules without cells. A high concentration of tubulin dimers and GTP are needed for in vitro polymerization of microtubules. A high concentration of GTP-bound heterodimers is needed to promote nucleation of tubulin subunits in a test tube.

Describe how a microtubule cycles between growing and shrinking in a test tube.

GTP-bound heterodimers have a straight shape so they can create straight protofilaments at the plus end. This GTP cap is very stable because the straight shape of the heterodimers maximizes the number of lateral interactions between the globular-shaped beta and alpha subunits in the heterodimers. This GTP cap is maintained as long as the rate of addition of heterodimers to the plus end of the microtubule is faster compared to the rate of GTP hydrolysis of the beta subunits in the heterodimers. As the microtubule grows the local concentration of GTP-bound heterodimers decreases so the rate of addition eventually slows down. The slower rate of adding GTP-bound heterodimers to the plus end promotes the loss of a GTP cap because most of the heterodimers in the plus end have GDP bound to the beta subunit. When a large number of heterodimers are bound to GDP at the plus end they have a crooked shape (not straight) which minimizes lateral interactions between the globular-shaped beta and alpha subunits in heterodimers. Reducing the lateral contacts promotes dissociation of heterodimers from the plus end (microtubule shrinking). As heterodimers dissociate that automatically exchange GTP for GDP, so the concentration of GTP-bound heterodimers starts to increase. When the concentration of GTP-bound heterodimers is high enough the microtubule is able to form a GTP cap and begin growing again.

What is the difference between growing microtubules in a test tube versus growing microtubules in a living cell?

In vitro polymerization requires a very high concentration of tubulin dimers and GTP because this facilitates spontaneous nucleation of dimers in a test tube. The cytosol has a high concentration of GTP but a low concentration of tubulin dimers so spontaneous nucleation cannot occur in the cytosol. Cells are able to nucleate tubulin dimers at a low concentration because they have tubulin-binding proteins.

What are gamma-tubulin ring complexes? Where are they located? What is their function?

Gamma-tubulin ring complexes represent thirteen different gamma-tubulin proteins that are connected together in a circle and located on the surface of a centrosome. Gamma-tubulin ring complexes are nucleation sites for growth of one microtubule in a cell because each gamma-tubulin protein can bind to an alpha tubulin subunit in a tubulin heterodimer. The binding of thirteen heterodimers to thirteen gamma-tubulin arranged in a ring promotes the formation of 13 protofilaments to create a hollow cylinder. Because the concentration of tubulin dimers is very low within cells, gamma-tubulin ring complexes on centrosomes are the primary site for growth of microtubules.

When cells make microtubules, what determines the number of microtubules? What determines the rate of growth?

he number of nucleation sites (gamma-tubulin ring complexes) in a centrosome determines the number of microtubules in the cell. The concentration of GTP-bound heterodimers in the cytosol determines the rate of growth. As microtubules grow inside cell the local concentration of GTP-bound heterodimers changes so dynamic instability is also observed in cells.

. Describe the function of plus-end capping proteins within cells.

Plus-end capping proteins are used by cells to stabilize microtubules so they will not be constantly growing and shrinking. These proteins stabilize tubulin dimers at the plus end of a microtubule and prevent dissociation of the subunits. This typically occurs by stabilizing interactions between GDP-tubulin at plus end or preventing tubulin dimers at plus end from hydrolyzing the bound GTP. Plus end capping proteins are important for intracellular transport and function of the flagellum because these processes require stabilized microtubules.

Describe the functions of dynein and kinesin. How are they similar? How are they different? How do they contribute to protein sorting?

Kinesin and dynein are both microtubule-binding proteins. They are also both motor proteins (they can bind to cargo and transport the cargo in a specific direction).

Kinesin is a motor protein that carries organelles and vesicles toward the plus end of microtubules.

Dynein is a motor protein that carries organelles and vesicles toward the minus end of microtubules.

Explain why kinesin, microtubules, and plus-end capping proteins are important for neuronal signaling.

Neurons use microtubules to transport synaptic vesicles and macromolecules between the cell body and nerve terminal. Microtubules produced from the centrosome near the nucleus of the neuron extend to nerve terminal and are bound by plus-end capping proteins to stabilize the microtubule and prevent rapid growing and shrinking (dynamic instability). Kinesin transports vesicles from the cell body to the nerve terminal (outward transport) which can be used to transport neurotransmitters synthesized in the cell body to the nerve terminal so they can be released upon arrival of an action potential.

What are some important functions of actin filaments within a cell?

Actin helps with cell shape (microvilli increase cell surface area), cell motility (crawling on a solid surface), muscle contraction (myosin binds to actin filaments), and cell division (contractile ring for cytokinesis)

Describe the subunits and protofilaments in actin. How are they used to assemble actin filaments? How many protofilaments are in an actin filament? What is the plus and minus end of an actin filament?

The subunits for actin filaments are globular monomers that are asymmetrical. One protofilament is created when monomers are stacked on top of each other in the same orientation (barbed end on top, pointed end on bottom). The protofilament has polarized ends because the barbed end of a monomer is exposed on one end and the pointed end of a monomer is exposed on the other end. Two protofilaments combine to form a thin helical chain with a plus end (barbed end) and minus end (pointed end).

Describe the significance of ATP hydrolysis in an actin filament.

When the actin monomer is not incorporated into an actin filament it is bound to ATP, but the rate of ATP hydrolysis is low. When the actin monomer becomes incorporated into a growing actin filament, the rate of ATP hydrolysis increases. ATP hydrolysis by actin monomers is important for growth and disassembly of actin filaments because it regulates the shape of actin monomers which influences lateral interactions with other actin monomers in the actin filament. ATP-bound monomer = lots of interactions with surrounding monomers (stable); ADP-bound monomer = fewer interactions with surrounding monomers (less stable).

Describe the significance of ATP hydrolysis in an actin filament.

When the actin monomer is not incorporated into an actin filament it is bound to ATP, but the rate of ATP hydrolysis is low. When the actin monomer becomes incorporated into a growing actin filament, the rate of ATP hydrolysis increases. ATP hydrolysis by actin monomers is important for growth and disassembly of actin filaments because it regulates the shape of actin monomers which influences lateral interactions with other actin monomers in the actin filament. ATP-bound monomer = lots of interactions with surrounding monomers (stable); ADP-bound monomer = fewer interactions with surrounding monomers (less stable).

. Describe the three steps of actin polymerization in a test tube: nucleation (lag phase), elongation (growth phase), and steady state (equilibrium phase).

Nucleation (lag phase) describes the time it takes for individual subunits to form an initial aggregate that can be used to grow a filament. Nucleation can only occur in a test tube (in vitro polymerization) if the concentration of subunits is very high. Cells use actin-binding proteins to promote nucleation.

Elongation (growth phase) occurs as subunits are added to the exposed plus ends of subunits in the initial aggregate. As subunits are added to the plus end to rapidly grow the filament the local concentration of subunits decreases.

Steady state (equilibrium phase) is reached when the rate of growth at the plus end matches the dissociation of subunits at the minus end.

Steps for in vitro polymerization:

• If concentration is high, an initial aggregate of monomers form (nucleation).

• Once nucleation occurs, there is a rapid growth of actin filaments (elongation).

• As concentration declines, filament reaches a steady state called treadmilling (rate of addition = rate of dissociation).

What happens when preformed actin fragments are added to a test tube with actin monomers and ATP?

The nucleation stage is bypassed, allowing faster polymerization of actin filaments because there is no lag phase.

What is treadmilling? How is it similar to dynamic instability? How is it different?

Treadmilling is the simultaneous gain of monomers at the plus end and loss of monomers at the minus end. This occurs in actin filaments during the steady state or equilibrium phase of polymerization. Dynamic instability is the rapid switch from growth to shrinkage that is observed during the steady state of microtubule polymerization. The gain and loss of dimers primarily occur at the plus end in microtubules.

How do cells regulate actin polymerization?

The concentration of actin subunits inside the cytosol is very high, so cells regulate actin polymerization with actin-binding proteins to prevent spontaneous polymerization. Some actin-binding proteins (profilin and thymosin) bind to actin monomers that have not been incorporated into filament while other actin-binding proteins bind to actin filaments (Arp complex).

What is the function of the Arp complex?

The Arp complex nucleates two actin monomers with the plus ends pointing up so that they can be used to grow two vertical actin protofilaments. The Arp complex can also bind to a pre-existing actin filament to create a branched network when it nucleates a new filament.

What are the functions of profilin and thymosin in cells?

Profilin and thymosin regulate the ability of free actin monomers to incorporate into a growing actin filament. The cytosol always has a high concentration of actin monomers, but cells also maintain a high concentration of thymosin, so most actin monomers are bound to thymosin. When an actin monomer is bound to thymosin it cannot nucleate or attach to a growing end of a filament. Cells activate pre-existing profilin which can rapidly strip actin monomers away from thymosin because profilin can also bind to actin monomers, but an actin monomer cannot be bound to thymosin and profilin at the same time. When an actin monomer is bound to profilin it can attach to the plus end of a growing actin filament. This system allows cells to control when and where actin polymerization takes place in the cytosol. Without thymosin there could be spontaneous nucleation of actin in the cytosol.

Describe how the Arp complex, profilin, and thymosin contribute to the production of membrane protrusions.

The Arp complex promotes nucleation by binding to the pointed (minus) ends of two actin monomers so the barbed (pointed) ends are exposed. Profilin is activated so actin monomers are removed from thymosin and are now bound to profilin. Actin monomers that are bound to profilin can bind to plus ends of growing actin filaments. Because Arp complexes bind to pre-existing actin filaments as actin filaments are growing, branched networks of actin filaments are formed.

What is myosin II? What is the function of myosin II in human cells?

Myosin II is a motor protein (walks toward plus end of actin) that functions as a homodimer. It interacts with actin filaments and is important for muscle contraction.

. Describe the general structure of myosin II by explaining the roles of the head, neck, and tail domains.

Head: binds ATP and actin; has ATPase activity.

Neck: provides lever arm for movement; different myosin types have different amounts of light chains.

Tail: allows monomer subunits to dimerize and aggregate with other myosin dimers to form a myosin filament.

. What is unique about myosin II? Why is this unique characteristic important for its function in muscle cells?

Myosin II is the only myosin that can form bipolar myosin filaments. This allows the myosin heads to move in opposite directions when they interact with different actin filaments. The movement of myosin heads in opposite directions generates muscle contraction by minimizing the distance between Z discs in sarcomeres.

What is formin? How is formin similar to the Arp complex? How is it different?

Formin is an actin-binding protein that nucleates actin filaments, like the Arp complex. However, formin produces unbranched actin filaments and binds to the plus end of a growing actin filament. The Arp complex binds to the minus end of a growing actin filament and to another preexisting filaments to create a branched network actin filaments.

What is a sarcomere? Describe the structure of a sarcomere using the terms actin, myosin II, tropomodulin, Z disc, plus end, and minus end.

A sarcomere is a basic contractile unit of muscle composed of actin (thin) and myosin II (thick) filaments. The actin filaments in a sarcomere are capped with Z discs at the plus ends and capped with tropomodulin at the minus ends. Myosin II filaments are at the center of the sarcomere, and their heads move toward the plus ends of both actin filaments (toward Z discs), pulling the actin filaments inward and shortening the sarcomere.

Describe the functions of tropomyosin, troponin, and calcium in muscle contraction.

Tropomyosin: actin-binding protein in muscle cells that blocks myosin binding sites on actin filament in sarcomere.

Troponin complex: multi-protein complex that binds to actin, tropomyosin, and calcium; moves tropomyosin in response to calcium.

Calcium: when it enters the cytosol of the muscle cell it binds to troponin complexes which causes tropomyosin to move, myosin binding sites are exposed, and myosin binds to actin to initiate contraction (movement towards the plus ends of actin).

How does myosin use ATP to promote contraction in a sarcomere? Need to be able to describe this process in a sequence of steps.

1. Myosin head that is not bound to ATP or ADP is tightly interacting to actin monomer in actin filament.
2. ATP binds to myosin head which causes myosin head to change shapes and detach from the actin monomer.
3. Myosin head hydrolyzes ATP which changes position of neck domain/lever arm (ADP and phosphate are still attached to the myosin head).
4. Myosin binds to weakly at new actin monomer (towards plus end) in actin filament.
5. ADP and phosphate are released from myosin head, and neck domain regains original shape. The return of the neck domain to its original position creates the power stroke which pulls the actin filament past the myosin filament; myosin head is tightly bound to actin monomer in actin filament without ATP or ADP.

What happens to a sarcomere during contraction?

The distance between the two Z discs in a sarcomere shortens during muscle contraction because two capped actin filaments slide past the myosin filament.

What is the relationship between a sarcomere, skeletal muscle cell, and myofibril?

Sarcomere → myofibril → skeletal muscle cell

Sarcomeres shorten when there is an influx of calcium in the cytosol of a muscle cell because myosin heads can bind to actin monomers. There are thousands of sarcomeres packed into myofibrils and myofibrils go from end-to-end within a striated muscle. When the thousands of sarcomeres shorten at the same time, they myofibrils shorten. When myofibrils shorten the shape of the muscle changes from long and skinny to short and compact. When millions of muscle cells become compact at the same time a muscle tissue contracts.

Comparison table (visible portion)

	Intermediate filaments	Microtubules	Actin filaments
Function in cell	Maintain cell structure, help with stability, and provide mechanical strength.	Important for segregating chromosomes during mitosis. Cell movement with flagella and cilia in liquid environments.	Changing the shape of the cell. Muscle contraction. Cell movement in a dry environment (cell crawling).

Function in cell (continued)	End to end within cells to provide strength.	Intracellular transport with kinesin (moves toward plus end) and dynein (moves toward minus end).	Makes contractile ring in cytokinesis.
Subunit description	Homodimers (coiled monomers). Homodimers are thin and elongated to maximize non-covalent interactions when they are used to build filaments.	Heterodimer with alpha and beta tubulin. Low concentration inside cell. Beta tubulin can hydrolyze GTP, alpha tubulin can bind to GTP but cannot hydrolyze.	Asymmetrical monomers. High concentration inside cell. Actin monomers bound to ATP are typically inactivated by thymosin so spontaneous polymerization cannot occur.
Protofilament description	Staggered tetramer. Subunits in antiparallel orientation make non-polar ends and increase stability.	Linear strand of heterodimers with same orientation in the subunits (beta on top, alpha on bottom). Same orientation creates plus end and minus end.	Linear strands of monomers in the same orientation. Pointed end down to create minus end and barbed end up to create plus end.
Filament description	Eight protofilaments to make a stable rope. Length of rope can be adjusted to fit cell’s needs.	13 protofilaments arranged in circle to make rigid hollow cylinder.	Two protofilaments create thin helix. Cells can make parallel bundles of actin filaments or make a branched network of actin filaments.
Nucleotide hydrolysis	N/A	GTP	ATP
Nucleation factors	N/A	Gamma tubulin in the centrosome. Gamma tubulin is in a ring complex called the gamma-tubulin ring complex. There are always 13 gamma tubulins in a gamma tubulin ring complex to make 13 protofilaments.	Formin creates parallel bundles that are observed in the sarcomere. Arp complex makes branched network and binds to minus end and to a preexisting actin filament.

Describe epithelial tissues. What is their function in multicellular organisms? How do they perform this function? Provide some examples of epithelial tissues.

Epithelial tissues are composed of epithelial cells that are tightly bound by cell-cell junctions with very little extracellular matrix between them. The lack of space in between the cells makes the epithelium an avascular tissue which means there are no blood vessels. They function as protective barriers for both external and internal surfaces in the body. Epithelial cells use tight junctions to prevent leakage and are anchored to the basal lamina with cell-matrix junctions to give epithelial tissues the ability to withstand mechanical stress.

What is the basal lamina? Where is it located and what is its purpose in cell adhesion?

The basal lamina is a specialized extracellular matrix on the basal (bottom) side of epithelial tissues. The basal lamina is composed of specific proteins that that provide strength and binding sites for cell-matrix junctions with epithelial cells.

Describe connective tissues. Where are they in relation to epithelial tissues? How are they different from epithelial tissues?

Connective tissues have cells that are sparsely distributed with very little cell-cell junctions and lots of extracellular matrix. The lack of cell-cell junctions creates space for blood vessels so connective tissues are typically vascular tissues that have lots of blood vessels.

Do epithelial tissues use cell-cell junctions, cell-matrix junctions, or both?

Epithelial tissues use both. Epithelial tissues use cell-cell junctions between epithelial cells and cell-matrix junctions between epithelial cells and the basal lamina.

What are anchoring junctions? Are they used for cell-cell junctions, cell-matrix junctions, or both?

Anchoring junctions are cell-cell or cell-matrix junctions that use the cytoskeleton and transmembrane proteins to attach cells to extracellular structures (adjacent cells or the extracellular matrix).

. Describe the following junctions and indicate if it is a cell-cell or cell-matrix junction: tight junction, adherens junction, desmosome, gap junctions, and hemidesmosome. You should also indicate if it is an anchoring junction or not.

Tight junctions: cell-cell junction, not anchoring; they prevent liquid from leaking through epithelial tissues and restrict the movement of membrane proteins.

Adherens junctions: cell-cell junction, anchoring; use actin and cadherin to connect adjacent cells.

Desmosomes: cell-cell junction, anchoring; use intermediate filaments and cadherin to connect adjacent cells.

Gap junctions: cell-cell junction, not anchoring; use connexin to link cytoplasm of adjacent cells for cell communication.

Hemidesmosomes: cell-matrix junction, anchoring; use integrin and intermediate filaments to attach cell to the extracellular matrix (basal lamina).

. What are transmembrane adhesion proteins? What does the intracellular domain do in a transmembrane adhesion protein? What does the extracellular domain do in a transmembrane adhesion protein?

trransmembrane adhesion proteins are membrane proteins observed in epithelial cells that span the plasma membrane. The intracellular domain of transmembrane adhesion proteins indirectly binds to the cytoskeleton, and the extracellular domain of a transmembrane adhesion protein binds to a structure outside the cell (another transmembrane adhesion protein or an extracellular matrix protein).

Describe the difference between homophilic and heterophilic binding. Provide an example for each type of binding that was discussed in class.

Homophilic binding describes the extracellular domain of a transmembrane adhesion protein binding to the extracellular domain of an identical transmembrane adhesion protein (e.g., cadherin binding to cadherin). Heterophilic binding involves two different proteins interacting outside of the cell. For instance, the extracellular domain of integrin (a transmembrane adhesion protein) binds to collagen (an extracellular matrix protein in the basal lamina).

Describe the function of adaptor proteins in cell adhesion.

Adaptor proteins link intracellular domains of transmembrane adhesion proteins to the cytoskeleton (actin filaments or intermediate filaments).

. Describe the function of cadherins. How do they contribute to cell adhesion? How does calcium contribute to the function of cadherins?

Cadherins are transmembrane adhesion proteins observed in epithelial cells that mediate cell-cell junctions in epithelial tissues. They bind to actin filaments (adherens junctions) or intermediate filaments (desmosomes) using their intracellular domain and adaptor proteins. They use their extracellular domain to bind to cadherins on adjacent cells (homophilic binding). Cells maintain a high extracellular concentration (low intracellular) of calcium ions. Cadherins use the extracellular calcium (Ca²⁺) ions to make stable extracellular domains. These proteins shapes (supported by calcium ions) use lateral interactions with other cadherins to make strong bonds between cells.

Why are cadherins important for development? Describe two experiments that demonstrated the importance of cadherins in development.

Cadherins are important for development because they allow cells of the same tissue to preferentially adhere to one another during the early stages of development. For instance, in one experiment calcium was removed from the media (extracellular fluid) that was supporting frog embryo development. Without calcium, cadherins from different tissues were unable to bind to each other using homophilic binding and cells in the frog embryo were in a random mixture instead of defined tissues. As soon as calcium was added back to the media, the cells in frog embryos reassembled into an organized structure because of homophilic binding. Another experiment used two different types of cultured cells (cells from a multicellular organism that are grown in a petri dish with media). These cells were initially grown in separate petri dishes, but when both cell types were dissociated and mixed together in the same petri dish they sorted out in the petri dish. This means specific cell types aggregated together and because of homophilic binding between different cadherin proteins. For multicellular organisms, expression of different types of cadherin (E-cadherin and N-cadherin) in different cell types during the early stages of development allows specific cell types to stay attached as the organism grows.

What is the role of catenin in cell adhesion?

Catenin is an adaptor protein that links cadherin to actin filament in epithelial cells. Cadherins connect to the cytoskeleton because they form cell-cell anchoring junctions, but they do not bind directly to the cytoskeleton.

What is the role of actin filaments in adherens junctions? What is the association between adherens junctions and the Arp complex?

The plasma membranes of adjacent cells must make physical contact to create adherens junctions between them; membrane protrusion from actin polymerization allow adjacent cells to make contact. Two cells use the Arp complex to nucleate actin polymerization at the plasma membrane. Because the Arp complex creates a branched network of actin filaments the plasma membrane is pushed forward creating a membrane protrusion. When the plasma membranes between adjacent cells make contact cadherins and catenins are recruited to the point of contact. When cadherins, catenins, and actin filaments are in the same location they can be connected to create an adherens junction.

What is an adhesion belt?

It is a continuous band of adherens junctions between adjacent epithelial cells that is located below the tight junctions.

. How are keratins similar to nuclear lamins? How are they different?

Keratins and nuclear lamins are both subunits (elongated proteins that form homodimers) for intermediate filaments. Nuclear lamins are used to form the nuclear lamina in the nucleus that provides structural support for the nucleus. Keratins are used by epithelial cells to contribute to desmosomes for cell adhesion. Keratins can bind to adaptor proteins that are attached to cadherins in the cytosol of epithelial cells.

Compare desmosomes and adherens junctions. How are they similar? How are they different?

Desmosomes and adherens junctions are both cell-cell anchoring junctions. However, adherens junctions are more dynamic because they are linked actin filaments and because cell signals are used to regulate assembly and disassembly. Desmosomes are more stable due to their connection to intermediate filaments and because they are observed in tissues that experience mechanical stress such as cardiac muscle tissue.

Describe the association between connexins and gap junctions. How are connexins similar to cadherins? How are they different?

Connexins are the transmembrane proteins that form gap junctions in epithelial tissues. Connexins are also transmembrane proteins and used for cell-cell junctions like cadherin. However, connexin is not a transmembrane adhesion protein because it does not interact with the cytoskeleton and gap junctions are not anchoring junctions. Connexins use homophilic and heterophilic binding to create channels of various sizes because gap junctions allow the movement of small molecules between cells. Cadherins use homophilic binding to form anchoring junctions.

. What is the extracellular matrix? How is the extracellular matrix produced in multicellular organisms?

The extracellular matrix is a complex mixture of glycosylated proteins and polysaccharides that is secreted by cells within the extracellular matrix. It is produced when cells synthesize ECM components in the rough ER and Golgi and then secrete them into the extracellular space, where they assemble into a matrix. This means that all protein components of the extracellular matrix had an ERSS (signal sequence at the N-terminus) during translation that was cleaved at the end of translation. The proteins in the extracellular matrix were secreted using the constitutive secretory pathway.

Describe the functions glycosaminoglycans (GAGs), proteoglycans, collagen, and fibronectin in the extracellular matrix.

GAGs are repeating disaccharides that occupy space in the extracellular fluid and form a “soft sponge” that can be compressed because polysaccharides cannot form compact structures like polypeptide chains but are still very hydrophilic. GAGs with attached polypeptide chains are called proteoglycans. Collagen is a fibrous protein secreted by cells that strengthens the extracellular matrix. It is a major component of skin and bone, and its structure is a triple helix. Fibronectin is a scaffolding protein that has many different binding sites for proteins in the extracellular matrix such as integrin and collagen. Because fibronectin can bind to different components in the extracellular matrix, it is involved with organizing the matrix.

How is the extracellular matrix able to create hard substances like bone and soft substances like cartilage?

The ability of the extracellular matrix to create hard or soft substances depends on its specific composition. When it is composed of many proteoglycans and not mineralized, it is flexible and softer. However, when the extracellular matrix has few proteoglycans and is mineralized it becomes hard bone.

What are integrins? How are integrins similar to cadherins, how are they different?

Integrins are transmembrane adhesion proteins that mediate cell-matrix anchoring junctions. Integrins are similar to cadherins as they are both transmembrane adhesion proteins involved with anchoring junctions and both can attach to actin and intermediate filaments with adaptor proteins and the intracellular domain. However, integrins participate in heterophilic binding when interacting with extracellular proteins (such as fibronectin) whereas cadherins participate in homophilic binding with other cadherins in cell-cell junctions.

Describe how integrins, collagen, and fibronectin work together in a cell matrix junction.

n an actin-linked cell-matrix junction, integrin is attached to an actin filament using its intracellular domain and an adaptor protein. The extracellular domain of integrin binds to fibronectin. Fibronectin is a scaffolding protein with many different binding sites, so it is able to connect integrin to collagen.

Compare hemidesmosomes and actin-linked cell-matrix junctions. How are they similar? How are they different?

Both facilitate cell-matrix anchoring junctions using integrin and extracellular matrix proteins.

Hemidesmosome: integrin is indirectly attached to intermediate filaments inside the cell and binds to laminin on the

Describe how integrins, collagen, and fibronectin work together in a cell matrix junction.

In an actin-linked cell-matrix junction, integrin is attached to an actin filament using its intracellular domain and an adaptor protein. The extracellular domain of integrin binds to fibronectin. Fibronectin is a scaffolding protein with many different binding sites, so it is able to connect integrin to collagen.

Compare hemidesmosomes and actin-linked cell-matrix junctions. How are they similar? How are they different?

Both facilitate cell-matrix anchoring junctions using integrin and extracellular matrix proteins.

Hemidesmosome: integrin is indirectly attached to intermediate filaments inside the cell and binds to laminin on the outside of the cell; laminin binds to collagen.

Actin-linked cell-matrix junctions: integrin is indirectly attached to actin filaments inside the cell and binds to fibronectin on the outside of the cell; fibronectin binds to collagen.

. What is signal transduction? What is the purpose of signal transduction within cells?

Signal transduction refers to converting one form of a signal to another. Signal transduction within cells allows cells to convert an extracellular signal into an intracellular signal that can trigger a specific cellular response.

How does one signal like acetylcholine promote different responses in the same organism?

Acetylcholine can promote different responses in the same organism because different cell types express different receptors. The cellular response to an extracellular signal like acetylcholine is defined by the receptor on the target cell and the downstream signal transduction pathway. Receptors on different cell types can bind to acetylcholine and use different pathways to generate different responses. For instance, a heart pacemaker cell receptor could lead to a decrease rate of firing when exposed to acetylcholine, while a salivary gland cell receptor could lead to secretion when exposed to acetylcholine.

. Describe how an intracellular receptor initiates a response within a cell. Include nuclear localization signal and estrogen receptor in your description.

Signaling molecules can either diffuse through plasma membrane or bind to receptors embedded in the plasma membrane to initiate signal transduction. Small or hydrophobic signals diffuse across membrane and bind to an intracellular receptor. Intracellular receptors often localize to the nucleus and regulate gene expression. For example, when the estrogen hormone is released into the bloodstream it can diffuse through the plasma membrane of many different cell types and bind to an estrogen receptor in the cytosol. The binding of estrogen hormone to the estrogen receptor changes the shape of the receptor which exposes a nuclear localization signal (NLS). The NLS on the estrogen receptor is recognized by a nuclear import receptor so the estrogen receptor is imported into the nucleus where it changes gene expression to initiate a cellular response.

Why do some signaling molecules use an intracellular receptor and other signaling molecules require a cell-surface receptor for cell signaling?

Small or hydrophobic signaling molecules (like the estrogen hormone) use an intracellular receptor to trigger a response because they can diffuse through the plasma membrane. Large or hydrophilic molecules (like growth factors) have to bind to a cell-surface receptor because they cannot diffuse through the plasma membrane to trigger a cellular response.

Identify and describe the three steps of cell signaling that are used when the extracellular signal is large and hydrophilic.

• Reception: Large or hydrophilic signaling molecules cannot diffuse through the plasma membrane so they require a cell-surface receptor so the signal can bind to the extracellular domain of the receptor.

• Transduction: The transmembrane receptor converts the extracellular signal into an intracellular signal. Intracellular signaling molecules are used to transduce and amplify the signal to activate a large number of intracellular molecules.

• Response: The small intracellular molecules eventually alter activity of effector proteins that have a direct effect on behavior of target cells.

What is primary transduction?

Primary transduction is the process in which a cell-surface receptor (a transmembrane protein) converts an extracellular signal into an intracellular signal.

7. Why are molecular switches important for cell signaling?

Molecular switches are important for signal transduction because they allow intracellular signaling proteins to switch between active and inactive states. This enables signaling pathways to be quickly turned on when an extracellular signal arrives and reset after the extracellular signal leaves and the cellular response is no longer needed.

Provide some examples of molecular switches that are used in cell signaling and explain why they are considered a molecular switch

RTKs and kinases in a kinase cascade are molecular switches because they use phosphorylation and dephosphorylation to activate and inactivate cell signaling pathways. G-proteins and Ras are molecular switches because they use GTP binding and GTP hydrolysis to activate and inactivate cell signaling pathways.

What are effector proteins in cell signaling? What is an example of an effector protein that is often used in cell signaling and is the focus for this course?

Effector proteins are proteins that have a direct effect on physical and chemical changes within a cell. For this course, we are focusing on how the activation of transcription factors from cell signaling pathways alter gene expression profiles in response to extracellular signals.

10. What are enzyme-coupled receptors?

Enzyme-couple receptors are transmembrane proteins that can act as intracellular enzymes when stimulated by an extracellular signaling molecule.

. What is the function of a protein kinase? Why are protein kinases important for enzyme-coupled receptors?

Protein kinases typically phosphorylate the side chain of one amino acid in a polypeptide chain. The addition of a phosphate group to an amino acid side chain changes the shape of the protein and therefore its function. The enzyme in enzyme-coupled receptors is typically a protein kinase. When an extracellular signal binds to an extracellular domain of an enzyme-coupled receptor, an intracellular kinase is activated (primary transduction).

What are receptor tyrosine kinases (RTKs)? How are RTKs activated? Describe signal transduction with RTKs.

Receptor tyrosine kinases (RTKs) are a type of enzyme-coupled receptor that function as intracellular protein kinases that phosphorylate tyrosine side chains on proteins inside the cell. Extracellular signaling molecules that bind to RTKs promote dimerization of RTKs for activation. When two RTKs dimerize (pair up), the close proximity of intracellular kinase domains promotes trans-autophosphorylation (tyrosine residues on the intracellular domain of RTKs are phosphorylated). The phosphorylated tyrosines on the activated receptors are docking sites for intracellular signaling proteins. These intracellular signaling molecules continue the process of signal transduction by allowing the signal to leave the receptor so it can be amplified and travel to the nucleus so gene expression can be altered

What is the epidermal growth factor receptor (EGFR)? What is the ligand for EGFR?

the epidermal growth factor receptor (EGFR) is a type of receptor tyrosine kinase (RTK) that is inactive when it exists as a single monomer. When the ligand, epidermal growth factor (EGF), binds to EGFR, it causes two EGFRs to dimerize (pair up). Dimerization of EGFRs activates the intracellular kinase domains, allowing each EGFR to phosphorylate tyrosine residues on the other (trans-autophosphorylation). These phosphorylated tyrosines then serve as docking sites for intracellular signaling proteins (Grb2 and Sos).

Describe the function of Grb2 and Sos in EGFR signaling.

Grb2 is a scaffolding protein with SH2 and SH3 domains. The SH2 domain of Grb2 binds to phosphorylated tyrosine residues on EGFRs. The SH3 domain on the same Grb2 protein binds to an Sos protein which has a Ras GEF domain. Binding of Sos to Grb2 brings the Ras GEF domain in close proximity to the plasma membrane. Ras is covalently attached to a membrane lipid on the cytosolic side so eventually the Ras GEF domain provided by Sos interacts with Ras. When Ras GEF interacts with Ras, the bound GDP is exchanged for GTP, activates Ras and allows the intracellular signal to leave the RTK.

What is Ras? Describe the function of Ras in EGFR signaling.

Ras is a monomeric GTP-binding protein that is covalently attached to a membrane lipid on the cytosolic side. Ras relays intracellular signals from RTKs to a kinase cascade.

What happens when a signal molecule binds to an EGFR? How does signal transduction occur? Use EGF, Ras, Raf, transcription factor, EGFR, and MAP kinase (aka Erk) in your explanation.

In EGFR signaling, when EGF binds to EGFR and the receptor becomes phosphorylated, Grb2 binds to phosphorylated tyrosine residues on EGFR using its SH2 domain. SH3 domain in Grb2 then binds to Sos which has an attached Ras-GEF domain that induces Ras to exchange GDP for GTP. Activated Ras binds and activates Raf (a protein kinase), which in turn activates Mek and then Erk in a kinase cascade. Erk activates a transcription factor which changes gene expression (cellular response).

What is the cellular response to activation of the EGFR signaling pathway?

Activate the cell cycle and promote cell division (cell growth) by promoting the expression of cell cycle genes.

Why are mutations at EGFR genes advantageous for cancer cells?

If a point mutation occurs in EGFR, the receptor can become constitutively active even when EGF is not present. When EGFR is always activated, the cell is continuously driven to divide, leading to uncontrolled cell division. This gives cancer cells a growth and survival advantage, leading to tumor development.

. What is a tyrosine kinase inhibitor (TKI)?

Tyrosine kinase inhibitors (TKIs) are inhibitors that are used to treat cancer patients that have point mutations in EGFR genes. TKIs will bind to the intracellular domains (kinase domains) of EGFRs preventing phosphorylation of tyrosine and ultimately preventing the activation of signaling pathways that promote cell growth and division. In cancer cells with EGFR mutations, TKIs help block uncontrolled cell division and limit tumor growth.

Need to be familiar with using two different antibodies to analyze the RTKs in a cell signaling pathway. One antibody analyzes total protein. A different antibody analyzes activated protein. How are these antibodies analyzing different versions of the same protein? Why are these antibodies used to measure the same protein? This topic was covered in HW6 and discussion activity 10.

The antibody that measures total protein binds to a protein structure that is not phosphorylated when the cell signaling pathway is activated. This antibody is used to measure the amount of both phosphorylated and unphosphorylated protein (total protein) in one sample. The antibody that analyzes activated protein can only bind to a tyrosine residue in the intracellular domain of the RTK if it has been phosphorylated. Using these two antibodies allow scientists to determine if a decrease in activated (phosphorylated) RTK is due to a decrease in total protein or a higher percentage of unphosphorylated protein.

What are G-protein coupled receptors (GPCRs)? What is a heterotrimeric GTP-binding protein?

G-protein-coupled receptors (GPCRs) are seven-pass transmembrane receptors that are coupled to a G-protein for primary transduction. The G-protein is a heterotrimeric GTP-binding protein with three subunits: alpha, beta, and gamma. When the GPCR is not bound to its extracellular signal, the alpha subunit is bound to GDP and associated with the beta and gamma subunits. When an extracellular signal binds to a GPCR, the intracellular domain of the receptor changes shape which activates an intracellular GEF domain. The GEF domain on the intracellular domain of the receptor interacts with the G-protein because it is covalently attached to a lipid on the cytosolic side of the plasma membrane. The interaction between the intracellular domain of the receptor and the G-protein causes the alpha subunit to exchange GDP for GTP, which activates the G-protein. The GTP-bound alpha subunit then dissociates from the beta and gamma subunits and can interact with intracellular signaling molecules to trigger a response. The pathway will be active until the GTPase activity of the alpha subunit hydrolyzes bound GTP to GDP which causes the alpha subunit to bind to the beat and gamma subunits and shut down the signaling pathway.

What is happening to a GPCR that is not bound to a signal? How does signal transduction occur when a signal is detected by the receptor?

A GPCR that is not bound to a signal is inactive. The alpha subunit in the G-protein is bound to GDP and attached to the beta and gamma subunits. When an extracellular signal is bound to the receptor, it triggers activation of the GEF domainand GDP in the alpha subunit is replaced by GTP. The beta and gamma subunits dissociate from the alpha subunit and can be used to stimulate intracellular signaling molecules for signal transduction.

. What are second messengers? What are some examples of second messengers in GPCR signaling pathways?

Second messengers are intracellular signaling molecules (small molecule or ion) produced or activated in response to an extracellular signal. For instance, cAMP and calcium are secondary messengers that are produced when GPCRs are activated by an extracellular signal.

What is cyclic AMP (cAMP)? How is it produced? What is its role in GPCR signaling?

cAMP is a second messenger made from ATP by adenylyl cyclase (a transmembrane protein). It helps amplify the intracellular signal when a GPCR is activated because millions of molecules of cAMP are produced when an extracellular signal binds to a GPCR.

How is the activity of protein kinase A (PKA) regulated? What is its role in GPCR signaling?

When the GPCR signaling pathway is inactive, PKA activity is inhibited by regulatory subunits. PKA is activated by the production of cAMP from adenylyl cyclase regulatory subunits are removed from PKA when cAMP levels increase. Once activated, PKA phosphorylates/activates the CREB transcription factor which alters gene expression.

. Describe the signaling pathway with a Gs G-protein using the following terms: CREB transcription factor, adenylyl cyclase, cAMP, PKA, alpha subunit, beta subunit, and gamma subunit.

An extracellular signal binds to the extracellular domain of a GPCR. The GPCR changes shape and the intracellular domain of the GPCR exposes a GEF domain that induces the alpha subunit in the G-protein to exchange GDP for GTP. The activated alpha subunit (with bound GTP) dissociates from the beta and gamma subunits, and binds to adenylyl cyclase (a transmembrane protein). When the GTP-bound alpha subunit interacts with adenylyl cyclase cAMP is made from ATP. The increase in intracellular cAMP removes regulatory subunits that inhibit PKA activity. PKA phosphorylates the CREB transcription factor which alters gene expression in the cell.

How is the glucagon hormone different from the estrogen in terms of bypassing the plasma membrane?

Glucagon is a large hydrophilic peptide so it cannot diffuse through the lipid bilayer and requires a GPCR (cell-surface receptor) to transduce intracellular signals. Estrogen is a small, hydrophobic hormone so it freely diffuses through the plasma membrane and binds to an intracellular receptor which activates a cellular response.

Briefly describe the function of the glucagon hormone, glycogen phosphorylase, and glycogen synthase.

Glucagon is a hormone that is secreted by the pancreas when blood glucose is low, and it promotes the breakdown of stored glycogen in liver cells. Glycogen phosphorylase is a cytosolic enzyme in liver cells and promotes the release of glucose from stored glycogen. Glycogen synthase is a cytosolic enzyme that promotes the production of glycogen in liver cells.

. Describe glucagon signaling in liver cells using the following terms: glucagon hormone, PKA, GPCR, glycogen synthase, cAMP, alpha subunit, CREB transcription factor, glycogen phosphorylase, glycogen breakdown, glycogen synthesis, glucose synthesis.

Glucagon binds and activates a GPCR on the surface of a liver cell. Once activated, the alpha subunit in the G-protein exchanges GDP for GTP and dissociates from the beta and gamma subunits. The GTP-bound alpha subunit activates adenylyl cyclase. cAMP is produced by adenylyl cyclase which activates PKA by binding to regulatory subunits that inhibit the kinase domain of PKA. The activation of PKA in liver cells promotes three different changes. PKA indirectly activates glycogen phosphorylase (PYG), which converts glycogen to glucose. PKA phosphorylates serine residues on glycogen synthase (GYS) which inhibits its activity. This prevents the synthesis of more glycogen from the glucose that has been liberated by PYG. Finally, the slow response to the signaling pathway is that PKA phosphorylates and activates CREB. This causes gluconeogenesis or the production of new glucose in liver cells. All three of these responses work together to promote the release of glucose from liver cells and inhibit the production of glycogen in liver cells so that blood glucose levels increase.

Describe some similarities and differences between glucagon signaling and epinephrine signaling.

Glucagon signaling and epinephrine signaling are both GPCR pathways that involve adenylyl cyclase, cAMP, and PKA. They also have similar effects, activating glycogen breakdown, inhibiting glycogen synthesis, and glucose synthesis through activation of CREB. However, they use different signaling molecules: liver cells respond to glucagon and muscle cells respond to epinephrine. They also use different GPCRs to detect these different signals. The final difference is that different physiological cues promote the release of the different signals. Glucagon is released because of low blood-glucose levels so different cell types have enough energy for survival. Epinephrine is released when there is danger and the increase in blood-glucose levels is used for an extra amount of activity so the organisms can run (or fight) to survive.

What are some similarities and differences between a stimulatory G protein (Gₛ) and a Gq protein (Gq)?

Gs is a G-protein that activates cAMP and thus protein kinase A when the coupled receptor binds to a signal. Gq G-proteins trigger different cellular responses because their coupled receptors bind to different signals. When a Gq G-protein is activated it will activate phospholipase C beta to produce second messengers like IP3 and calcium, which then activate protein kinase C. Both Gs and Gq G-proteins are heterotrimeric, 7 pass transmembrane proteins that activate their alpha subunit by binding GTP.