Cytoskeleton & Cell Signaling

Question 1

Discuss the concept of a cytoskeleton.

Answer 1

The cytoskeleton is a dynamic intracellular structure (or set of structures) thatprovides cell shape, mechanical strength, the structures needed for locomotion, support for the plasma membrane, the scaffold for the spatial organization of organelles, and the means for intracellular transport of organelles and other cargo.The cytoskeleton is formed by three different families of proteins: microfilaments, microtubules, and intermediate filaments.

Question 2

Describe microtubule and intermediate filament cytoskeleton (their properties, their functional roles, and their protein composition).

Answer 2

Microtubule: Alpha/Beta tubulin dimer, tubular structure 25nm in diameter, used for movement (flagella, cilia), cell organization, movement of organelles, cell division.Intermediate filament: Complex rope, 10nm in diameter, made of vimentin, keratin, and neurofilaments, used for mechanical stability.

Question 3

Discuss cytoskeletal dynamics and the role of certain proteins and drugs in tubulin polymerization/depolymerization.

Answer 3

Colchicine inhibits MT polymerization. Vinblastine and vincristine also are MT polymerization blockers, derived from the Madagascar periwinkle (Vinca rosea).Paclitaxel (Taxol) was first isolated from the Pacific yew tree and also binds to MTs, but it stabilizes them, which causes tubule and tubulin aggregates. These compounds and their derivatives block mitosis and, thus, are of great interest for cancer treatment.

Question 4

Explain the concept of molecular motor.Explain the mechanisms of tubulin-based movement and intracellular transport.

Answer 4

Many organelles (vesicles, mitochondria) travel long distances within cells, using MTs as tracks”. This is possible in conjunction with microtubule motor proteins. These proteins can transform the energy from ATP hydrolysis into a ‘walking’ motion along MTs

Question 5

Discuss the role of microtubules in mitosis.

Answer 5

The mitotic spindle is constructed from MTs and associated proteins and serves to segregate the replicated chromosomes during mitosis.Three types of MTs can be distinguished: astral MTs” that radiate out from the centrosomes; “kinetochore MTs” that are attached to the kinetochore formed at the centromere of each duplicated chromosome; and “overlap MTs” that interdigitate at the equator of the spindle.In all cases

Question 6

What do cilia and flagella do?Where are they found?

Answer 6

Cilia and flagella are wide-spread, hair-like cellular appendages that have a uniform diameter and contain a MT core, the axoneme.Flagella are long and serve to propel sperm by their undulating motion.Cilia are shorter and tend to occur in large numbers on the apical surface of various epithelial cells, especially those of the respiratory tract. By beating with a whip-like motion (across the cell surface) they move fluids over the surfaces of cells.In the respiratory tract, this serves to move dust particles, bacteria and mucus towards the mouth for elimination.Primary cilia or monocilia occur singly in a great variety of cells and may or may not be motile. Non-motile primary cilia serve special sensory functions, as photoreceptors (outer segment), chemosensors (e.g., in olfactory neurons), or mechanosensors.

Question 7

What is an axoneme, its structure, and how does it move?

Answer 7

The axoneme can be 10-200nm long and consists of MTs and their associated proteins arranged as a ring of 9 special doublets (one complete plus one partial MT each) surrounding a pair of single MTs (9 + 2 array).Accessory proteins hold the array together, and axonemes are anchored in basal bodies.Axoneme bending results from the action of dynein. The motor action of dynein heads causes MTs to slide against one another. Because MTs are anchored to one another dynein action results in bending.Highly coordinated dynein activity in the axoneme causes flagellar waves or ciliary beating motion.

Question 8

Discuss the cytoskeleton in the context of disease processes.

Answer 8

Trouble with cilia can cause numerous diseases, one being polycystic kidney disease.Microtubules are an important drug target in chemotherapy.Trouble with intermediate filaments can cause Charcot Marie Tooth disease and epidermolysis. The list goes on…

Question 9

Discuss cytoskeletal dynamics and the role of certain proteins in actin filament formation, polymerization/depolymerization.

Answer 9

Arp2/3 + Formin start nucleation and seed the growth of actin. Arp2/3 makes tree like actin spindles while Formin seeds parallel spindles.Profilin (a protein) promotes polymerization on the plus end by converting ADP to ATP (in complex with G-actin).Capping proteins prevent the + end from growing and the - end from depolymerization.Cofilin severs the actin chain in half leading to depolymerization.

Question 10

Understand the role of actin cytoskeleton in epithelial cell polarity and discuss some diseases associated with that.

Answer 10

One of the most important functions of actin is anchoring proteins that are involved in Tight junction (TJ) and Adherens junction (AJ) formation. Decreased association of AJ proteins (cadherins and catenins) with actin leads to internalization of cadherins and loss of cell-cell adhesion, the step that is a prerequisite for epithelial-to-mesenchimal (EMT) transition and cancer formation.In addition, actin plays a key role in apical micorvilli formation. All actin plus-ends are anchored in the apical protein cap of the microvillus. Actin bundles are held together by the cross-linking proteins villin and fimbrin, and bundles are linked laterally to the plasma membrane by myosin-I.Loss of microvilli is observed in microvilli inclusion disease.

Question 11

Explain the concept of molecular motor. Explain the mechanism of actin-based organelle movement and muscle contraction.

Answer 11

Actin-binding motor proteins belong to the myosin family. Myosins (heavy chain) consist of a head region, which hyrdolyzes ATP to ADP. After hydrolyzing ATP the myosin head stretches towards and binds further down the + end of the actin tube. ATP then binds again and the cycle repeats. This is the power stroke.The ATP-driven walk of myosin heads along actin filaments results in the sliding-filament mechanism responsible for muscle contraction.Other non-conventional myosins, such as I and V, are associated with membranes and, thus, are involved in the F-actin-mediated movement of organelles.

Question 12

Discuss the concept and the key steps of cell movement.

Answer 12

During locomotion, amoeboid cells go through repeated cycles of protrusion (of lamellipodia, filopodia), attachment (of these protrusions), traction (to pull the cell forward), and detachment (of adhesion toward the rear).Tight coordination between actin cytoskeleton dynamics and cell adhesion is a prerequisite for migration.

Question 13

Discuss cell motility in the context of developmental and disease processes.

Answer 13

Lissencephaly: This is a severe defect of brain development resulting in a smooth cortical surface. Neuronal migration is a critical process for establishing the normal, complex cytoarchitecture of the brain. Loss-of-function of n-cofilin, an actin filament depolymerizing factor, results in lissencephaly and the associated severe mental retardation.Metastasis: Most of the terminal cancers are characterized by the spread of the tumors from the primary site (metastasis).

Question 14

Understand the role of actomyosin ring in cell division.

Answer 14

Actin plays a key role in cytokinesis. The formation and contraction of actomyosin ring drives the formation of the cleavage furrow and separation of the daughter cells.The site of actomyosin ring formation and the timing of its contraction are highly regulated events that determine the symmetry of cell division.

Question 15

Understand the mechanisms regulating the establishment and activation of actomyoisn ring.

Answer 15

If you know this please email ryan.roth@ucdenver.edu.Thanks!

Question 16

Understand the concept and know some examples of asymmetric cell division.

Answer 16

Examples of assymetric cell division: Red blood cells (no nucleus!). Generation of platelets. Spermatogonia. Epithelial cells.

Question 17

When is contact dependant signaling used?Synaptic?Endocrine?

Answer 17

Immune response (antigens)When you need a precise, fast, short lived response.When you need a slow, general, long term response.

Question 18

Know all the types of extracellular signaling molecules and how they relate to the forms of signaling.

Answer 18

Hydrophilic molecules are unable to cross plasma membrane. Instead they bind to cell-surface receptor on the target cell. Cell- surface receptors then in turn generate their own signal and send it to the target protein via help of intracellular signaling molecules. Can be stored intracellularly and released very quickly, usually used in nervous system.Hydrophobic molecules can cross plasma membrane. Those signaling molecules bind to intracellular receptors. Since hydrophobic signaling molecules are insoluble in water, in extracellular space they are usually bound to carrier protein. Cannot store it, no need for membrane receptor.Gases. Least understood. Most common is NO. Shares some properties with hydrophobic. Cannot be stored and can cross membrane easily, best for short distances.In addition, recent studies have identified a novel, non-canonical signaling pathways that mediates direct transfer of cellular materials between cells via exosomes. Exosomes are membrane bound organelles that are formed and secreted from signaling cell. Exosomes are then transported and uptaken by a target cell where the cargo is deposited.

Question 19

What are the receptor types and actions for hydrophobic signaling molecules?

Answer 19

Nuclear receptors live in the nucleus. Signaled by hydrophobic signalling molecules. In the absence of signaling molecules, nuclear receptors are usually bound to inhibitory protein(s) and are in inactive state.Hormone binding alters the conformation of receptor protein and causes the release of inhibitory protein. The nuclear receptor protein is then ready to bind to coactivator protein and induce transcription of selective group of genes that contain a receptor-binding element.

Question 20

What is the receptor type for hydrophilic signaling molecules? Actions?

Answer 20

Water-soluble signaling molecules bind to the cell-surface receptors on the target cells. These cell-surface receptors act as signal transducers by converting extracellular signaling molecule binding event into intracellular signal. Translate then amplify signal.

Question 21

What are the three types of cell surface receptors?

Answer 21

3 types: ion-channel-linked, G-protein-linked, and enzyme-linked.

Question 22

Please describe ion channel receptors.

Answer 22

Ion-channel-linked receptors are involved in rapid synaptic signaling between electrically exitable cells, such as neurons or muscle cells. Binding of an extracellular signaling molecule (also known as ligand) to the receptor transiently opens the channel and allows the ions to cross the membrane into the cell. That leads to change in membrane potential and results in exiting the cell.In the case of neurons, that results in release of neurotransmitter.In muscle cells, opening of the ion channel leads to contraction.

Question 23

Please describe G-protein linked receptors.

Answer 23

G-protein-linked receptors act indirectly to regulate the activity of separate membrane bound enzyme. The interaction between receptor and target enzyme is regulated by a trimeric GTP-binding protein (G protein). These proteins act as on and off switches to activate other enzymes.The activation or inhibiton of target enzyme changes the concentration of small intracellular signaling molecules.These molecules are usually nucleotide or lipid derivatives and are used to amplify the signal received by cell-surface receptor. Smallintracellular signaling molecules in turn regulate activity of the other intracellular signaling molecules, mainly protein kinases.The phosphorylation changes the function of the target protein and is usually the end of the signaling cascade.

Question 24

Please describe enzyme linked receptors.

Answer 24

Enzyme-linked receptors when activated by signaling molecule either function themselves as an enzyme or are directly linked to the enzyme that they regulate. Activation of enzyme-linked receptors initiate the signaling cascade that involve generation of small intracellular signaling molecules to amplify the signal and activation of protein kinases to phosphorylate target proteins.The cascade typically results in the activation of gene transcription in the nucleus.

Question 25

Understand the concept of signal integration.

Answer 25

One signal can have many functions and many signals can create the same function.

Question 26

List the other tools of the signaling pathways, including secondary signaling molecules.

Answer 26

Receptors for signaling molecules.Secondary messengers (further down the cascade).Secondary messengers include: Ca++, DAG, IP3, cAMP, NO.Protein modifications, protein-protein binding, GTP/GDP exchange.

Question 27

Describe the mechanisms for signal termination.

Answer 27

Extracellular signalling molecule: diffusion, inactivation, uptake into cellsReceptors: desensitization, reduction of binding, internalizationProtein binding and targeting: lack of the inducing stimulus, protein degradation.Phosphorylation and dephosphorylation of important proteins.Phosphodiesterases catalyze the breakdown of cAMP to AMP, which is not a signalling molecule

Question 28

What are the amplification pathways?

Answer 28

Amplification: positive feedback loops (Calcium triggered calcium release), but must be carefully coupled with negative feedback systems to avoid spiraling out of control. Another way is by signaling cascades. Amplification depends on how many downstream molecules are affected and how long the upstream molecule stays active.

Question 29

Identify nodes and modules in a signaling pathway, and evaluate the potential for crosstalk in signal transduction.

Answer 29

Nodes: Points within a network with mutliple inputs or outputs. Pathways can go through a node. Multiple pathways utilize the same players.Modules: process signals together. Includes signal protein complexes and feedback mechanisms. Are a unit that work together. Group helpful things together- like the stove and the fridge help you understand the kitchen in the system of the house.There is extensive potential for crosstalk between pathways in networks. Complex regulation occurs with relatively few molecules.