Potential Concept Qs

Question 1

Protein estimations

Answer 1

400 AA
40 kDa
5nm
Lifetime (human cells) - 2 days
E. coli - 106 proteins in cell

Question 2

Cell estimations

Answer 2

≥ 3 orders of magnitude larger than proteins
Nucleus
Human cells - 10um
Human genome - 1m
Lifetime - up to 4 orders of magnitude longer than typical protein

Question 3

Upregulation of protein expression

Answer 3

Hours to days

Question 4

mRNA lifetime

Answer 4

Half life (human cells) - hours

Question 5

How big is a cell?

Answer 5

E. coli - 1um
Yeast - 5um
Animal - up to 100um, ish

Question 6

How crowded is the cytoplasm?

Answer 6

E. coli - ~1010 C atoms per cell
~5nm between proteins

An E.coli is 1 um in diameter and 1um3 in volume but contains around 106 proteins. Proteins are on average 5nm in diameter and about 3 orders of magnitude smaller than a cell. The average distance between proteins is 5nm. This does not take into account molecules like mRNA, filamentous cytoskeletal elements, organelles, etc… This, the cytoplasm is a highly crowded and dynamic environment, with different molecules and structures moving and interacting with each other in complex ways.

Question 7

What causes diffusion?

Answer 7

Brownian motion - random movement of particles
Einstein - results from collision of atoms

Diffusion is caused by the kinetic energy of the molecules resulting in random Brownian motion. It is driven by the random thermal motion and collisions of molecules.

Maximizes entropy
Isolated system will move towards macroscopic state with the highest entropy

Question 8

What is a diffusion coefficient and what determines this value for a molecule?

Answer 8

Rate of diffusion is affected by the concentration gradient, membrane permeability, temperature, pressure, size & shape of the cell, presence of membrane proteins or other structures that can facilitate or inhibit diffusion. Diffusion occurs spontaneously and does not require energy input. It is driven by the random thermal motion of the molecules, which leads them to move from areas of high concentration to areas of lower concentration to achieve a uniform distribution of the molecules.

Question 9

Diffusion coefficient (D)

Answer 9

is a measure of the movement of molecules in a substance (or in and out of the cell) due to diffusion.
Factors that affect diffusion coefficient:
-Viscosity of cytoplasmic fluid
-Collisions with other molecules
-Binding affinity for other molecules

Question 10

Bacteria are small cells without membrane-bound organelles, but they are not just well-mizedbags of proteins. Describe two mechanisms bacteria use to control protein localization patterns.

Answer 10

Diffusion to capture:

Reaction-diffusion

Question 11

Diffusion to capture

Answer 11

the spatial patterning of a molecule depends on the spatial localization of its receptor, so based on pre-patterned receptors. Structure is a competition between enthalpy and entropy. With a low concentration of the loans, the entropic cost of binding to a receptor is high. While in a high concentration of a ligand, the entropic cost is low and the energy released in binding (enthalpy reward) is worth the entropic cost.

Question 12

Reaction-diffusion

Answer 12

mechanism in which spatiotemporal patterns emerge from the amplification of random fluctuation. They result in spontaneous pattern formation. For example, FtsZ is restricted to midplane by reaction-diffusion:
MinD dimerizes and binds to the membrane (ATPase binding to the plane); it recruits MinC, which prevents Ftsz binding to the membrane ⇒ MinE replaces MinC and binds to MinD ⇒ initiates hydrolysis reaction removing MinD+MinE off the membrane ⇒ creates oscillation of MinD and MinE from one pole to the other in the cell.

Question 13

Diffusion time equation

Answer 13

Τ=x^2 /6D

x is distance, D is diffusion coefficient

Question 14

Why might it be advantageous for neurons to perform local translation in the axon terminal?

Answer 14

Allows for neurotransmitter release: neurotransmitter release is a rapid process that needs the synthesis of new proteins on demand which local translation satisfies.

Lower energetic costs: doesn’t rely on the transport of the protein from the cell body which can be slow and energy-intensive and prevent ectopic presence of proteins in other parts of the cell during protein transport.

Allows subcellular localization of proteins: mRNAs can be targeted to different subcellular localizations using “address” information in their untargeted regions.

Time scale - depending on length of axon. Would take longer to diffuse from soma to axon than a protein’s half life

Question 15

What is kinesin?

Answer 15

Kinesin is a biological motor protein that converts the energy released by ATP hydrolysis to mechanical energy and moves via a power stroke (1um/second). Kinesin transports cargo such as proteins, vesicles, organelles, etc… along microtubules towards the positive end (Karry out). It has motor domains that bind to MT and a cargo carrying domain that binds to the cargo.

Question 16

In brief, how was kinesis discovered?

Answer 16

Ron Vale
Myosin-coated beads move along actin filaments (Spudich and Sheetz)
–> Is myosin responsible for active transport in axons?

Injected myosin-coated beads into squid axons
–> Control moved but myosin-coated didn’t

In vitro reconstitution
–> MTs, ATP, membrane organelles → no movement so membranes don’t have motor bounds;
–> MTs + ATP + membrane organelles + soluble proteins
Movement
–>MTs + ATP + soluble proteins
Movement

The motor protein bound to the glass in place of the cargo and moved the MTs. Then he used column chromatography to isolate the soluble proteins that helped MTs move.

In vitro motility assay
Column chromatography with soluble proteins
Discovered kinesin

Question 17

How is the microtubule network organized in interphase versus mitosis

Answer 17

During interphase the centrosome is located near the nucleus and microtubules extend outward to the cell periphery. The centrosome anchors the minus ends of the microtubules.
During mitosis, the duplicated centrosomes separate and microtubules reorganize to form the mitotic spindle. MTs emanate from two centrosomes on either side of the cell, their plus ends directed toward each other. Mts also emanate from chromosome kinetochores resulting in a biased search and capture.

Question 18

what are the molecular mechanisms underlying these for MT network in interphase vs mitosis organization patterns? - interphase mechanism

Answer 18

Mechanism for Interphase: microtubules are tubulin heterodimers that assemble into 13 protofilaments (MT tube-like structure). The underlying mechanism for interphase MT organization is dynamic instability. MT polymerizes when the rate of polymerization (assembling of tubulin heterodimers) outpaces the rate of GTP hydrolysis (called rescue with GTP cap). During stochastic fluctuation, if the rate of GTP hydrolysis catches up with the rate of polymerization → then you lose the GTP cap at the plus which leads to a catastrophic event.
–> Alpha-tubulin and Beta-tubulin bind to GTP, and GTP at the alpha-tubulin is trapped between two tubulin subunits, so GTP cannot be exchanged or hydrolyzed.

Question 19

what are the molecular mechanisms underlying these for MT network in interphase vs mitosis organization patterns? - mitosis mechanism

Answer 19

Mechanism for Mitosis: Microtubules polymerize from the centrosomes and from the chromosome. MT polymerization at the centrosome is facilitated by the dynamic instability described previously. MT polymerizes near chromosome chromosomes via molecular mechanism underlying biased search and capture involving RanGTP.
—- >There is a high local concentration of RanGEFs near the choromossme that activates RanGTPase near the chromosome. RanGAP diffuses around the cell, inactivating RanGTP (that has diffused away from the kinetochore region). This results in RanGTPase being activated only near the kinetochores (not necessarily on it). Active RanGTP near the chromosome recruits -TURC, which nucleated polymerization near the kinetochore. Thus MTs nucleate at the kinetochores, and dyneins ride along them. When the MTs from the kinetochore “bump” into MTs nucleated from the centrosome, dyneins walk towards the “negative end” which is the centrosome. This results in bundles of antiparallel MTs.

Question 20

Microtubules exhibit “dynamic instability”. What does that mean

Answer 20

Microtubule dynamic instability is driven by GTP hydrolysis. Mts rapidly grow with a GTP cap or GTP-tubulin dimers at their plus end, because they can form a straight protofilament. GTP hydrolysis causes a conformational change in the subunit and weakens the binding affinity in the polymer, causing the protofilament to become curved. So loss of the GTP cap results in rapid shrinkage.
Dynamic instability is also regulated by microtubule-associated proteins, such as MAPs that stabilize MTs and TIPS that link plus ends with other structures.

Dynamic instability refers to the ability of microtubules to assemble and disassemble (at a constant rate) at the plus end only. The continuous switch between growth and shrinkage is known as dynamic instability. In contrast, treadmilling occurs when one end polymerizes while the other end depolymerizes.

Question 21

how is MT dynamic instability important for chromosome segregation during mitosis?

Answer 21

Dynamic instability facilitates a biased search and capture. During mitosis, the plus end of MTs emanating from the centrosomes switch polymerizing to rapidly depolymerizing. After catastrophe, MTs re-grow in a different direction until it binds to a kinetochore which stabilizes the MT plus end (by dynein walking towards the centrosome along the MT emanate by the centrosome??). Mts keep growing and shrinking until all kinetochores are captured. This dynamic instability allows the MT to search the cell for kinetochores.

Question 22

Actin filaments interact to form larger structures in cells. Name at least two of these structures and briefly describe what determines whether the actin network forms one structure versus the other.

Answer 22

Filopodia (fcn as antennae to probe environment) formed by core of long, bundled actin filaments. Lamellipodia (cell locomotion) formed by cross-linked mesh of actin filaments.

Question 23

How does the actin polymerization motor contribute to cell migration, and how do migrating cells keep this motor running? Pt A

Answer 23

Actin polymerization drives treadmilling which generates a force that pushes against the cell membrane and drives cell motility. Actin density is graded so that there are more actin filaments at the center of the leading edge. Because actin density is highest, the force exerted by the membrane (due to entropic penalty with stretching the membrane) per filament is low and actin rapidly polymerizes and produces protrusive forces. Filament density decreases towards the cell sides, so the force back from the cell membrane per filament is high and polymerization is stalled, which creates retraction at the trailing edge. The subcellular differences in actin polymerization allow for overall forward movement.

Question 24

How does the actin polymerization motor contribute to cell migration, and how do migrating cells keep this motor running? pt B

Answer 24

Molecular clutch model explains how migrating cells keep the motor running. Adhesions bind to the actin network, which opposes the myosin pull and prevents the inward flow at the leading edge. This allows actin polymerization to drive protrusion on the cell membrane.

Question 25

What are nucleosomes, and how do they affect transcription?

Answer 25

Positively charged histones are electrostatically attracted to negatively charged DNA and tightly associate to form nucleosomes.
DNA winds around the histone complex 2.5 turns which is around 150 bps.
A transcription regulator will bind with 20 times less affinity if its cis-regulatory sequence is near the end of a nucleosome and 200-fold less affinity if its in the middle of a nucleosome.

Question 26

What is the histone code and how does it affect chromatin structure?

Answer 26

Histones contain N-terminus tails that stick out from the nucleosome. Histone code is a specific pattern of post-translational modification on their tail, including phosphorylation, methylation, and acetylation on lysine and serine residues can be phosphorylated.

The histone code affects chromatin structure as it determines if it would be heterochromatin or euchromatin which in turn regulates gene expression through transcriptional accessibility. The histone code features Lys modifications as a major mechanism for the regulation of chromatin accessibility, gene expression, and cellular growth.

Question 27

What are topologically-associated domains (TADs), and how are they formed?

Answer 27

TADs are self-interacting regions of DNA with distinct boundaries.
CTCF complex is an insulator protein complex (barriers against the spreading of heterochromatin) that forms a dimer and defines TADs boundaries. CTCF complexes bind at specific sites in the DNA. Cohesin is a motor protein that forms a ring around the DNA and moves along the DNA strand upon ATP hydrolysis. As it moves, it threads the DNA through the cohesin ring to form a DNA loop until it is blocked by CTCF bound to DNA. CTCF anchors and stabilizes DNA interactions within the loop. This loop is a TAD.

Question 28

How does Ran GTPase regulate protein transport into the nucleus?

Answer 28

GTPase-activating proteins (GAPs) promote hydrolysis of GTP to GDP on the Ran protein, which leads to the release of Ran from the nuclear transport receptor and the dissociation of the protein complex.
Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on the Ran protein, which leads to the re-association of Ran with the nuclear transport receptor and the formation of a protein complex.
In summary, Ran GTPase plays a central role in the regulation of protein transport in and out of the nucleus by interacting with nuclear transport receptors. IT does this through the regulated exchange of GDP for GTP on the Ran protein, which is facilitated by GTPase activating proteins (GAPs) and Guanine nucleotide exchange factors (GEFs).
During nuclear import, an importin-α/β heterodimer forms a complex with cytoplasmic proteins tagged with a nuclear localization sequence (NLS) and transports the proteins into the nucleus. Ran-GTP binds to importin-β to dissociate the complex, releasing the transported protein.
During nuclear export, Ran-GTP binds and forms a complex with exportin and the protein to be transported out of the nucleus. Once in the cytoplasm, Ran-GAPs hydrolyze Ran-GTP to Ran-GDP which dissociates the complex releasing the transported molecule.

Question 29

How are proteins transported into the endoplasmic reticulum?

Answer 29

Overall, the process of protein transport into the ER involves the synthesis of the protein on the ribosome, the recognition of the protein by the signal recognition particle (SRP), and the transport of the protein across the membrane of the ER by the translocon.

an SRP (signal recognition protein) binds to an ER signal on the protein and the correlating ribosomes, pausing translation. The SRP-ribosome complex binds to an SRP receptor in the ER membrane. The SRP receptor directs the complex to a protein translocator in the ER lumen. The remaining SRP + SRP receptor is then recycled. The protein translocator inserts the polypeptide chain produced by the ribosome into the membrane to continue the rest of translocation directly into the ER.

Question 30

How are secreted proteins trafficked to the outer surface of cells? Start from the cytosol.

Answer 30

Secreted proteins are translated in the cytosol and transported to the ER. From the ER, they are trafficked to the Golgi through vesicles that bud off from ER and fuse with the Golgi membrane. They travel through the Golgi and bud off into secretory vesicles. The secretory vesicles then exocytose and fuse with the plasma membrane, secreting the proteins. The exocytosis is regulated by calcium which binds to Synaptotagmins. Upon the calcium signal, Synpatotagmins release complexin that was blocking the complete fusion of the primed vesicle.

Question 31

How are secreted proteins trafficked to the outer surface of cells? Start from the cytosol. - greater detail

Answer 31

Starts from synthesis in the cytosol. Secreted proteins are synthesized on ribosomes in the cytosol. As the protein is being synthesized, it is directed towards the endoplasmic reticulum (ER) by the signal recognition particle (SRP).
Transport to the ER. THe SRP recognizes a signal sequence on the newly synthesized protein and helps to direct it to the translocon which is a protein complex that spans the membrane of the ER. Once the protein reaches the translocon, it is folded into its correct conformation and then transported across the membrane into the lumen, or interior, of the ER.
Quality control. Once the protein is inside the lumen of the ER, it is subjected to a quality control mechanism that ensures it is properly folded and functional. If the protein is not properly folded or is not functional, it may be targeted for degradation or modification.
Transport to the Golgi apparatus. Proteins that pass quality control are transported from the ER to the Golgi apparatus, a membrane -bound organelle that is involved in sorting and modifying proteins. The Golgi apparatus modifies the proteins by adding carbohydrates or lipid groups, or by removing or adding amino acids.
Transport to the plasma membrane. Once the protein has been modified in the Golgi apparatus, it is transported to the plasma membrane, which is the outer membrane of the cell. Secreted proteins are typically transported to the plasma membrane in vesicles, which are small (100 nm), membrane-bound structures that contain the protein. The vesicles fuse with the plasma membrane and release the protein to the outside of the cell.
Overall, the process of transporting secreted proteins to the outer surface of cells involves several steps, including synthesis, transport to the ER, quality control, transport to the Golgi apparatus, and transport to the plasma membrane.

Question 32

What are phosphoinositides, and how do they contribute to cargo and target selectivity during vesicular trafficking?

Answer 32

Phosphoinositides (PIPs) are a family of acidic phospholipids in cell membranes. Specific PIPs mark different organelles and their membrane domains. The type of PIP depends on phosphorylation by kinases and dephosphorylation by phosphatases.

Vesicle formation involves adaptor proteins. Specific adapter protiens bind to specific PIPs. This binding causes a conformational change in the adaptor protein which exposes the binding sites for a specific cargo receptor that binds to a specific cargo protein. So the adaptor protein must be bound to its specific cargo receptor and the specific PIP to induce membrane curvature and vesicle budding. This allows the vesicles to only take up specific cargo proteins when budding.

Vesicle fusion involves Rab proteins which are specific GTPase localized on a specific organelle membrane. Active Rab binds to adaptor proteins which are linked to PIP. They are part of a specialized membrane patch that is specific to the target membrane. They both bind to specific Rab effector proteins, such as lettering proteins that tether the vesicle to the target membrane to fuse.

Question 33

How does the SNARE complex mediate vesicle fusion with a target membrane?

Answer 33

The SNARE (SNAP receptors) complex is a group of proteins that play a key role in mediating vesicle fusion with target membranes. It is composed of four SNARE proteins: two vesicle-associated SNAREs (v-SNAREs | synaptobrevin) and two target membrane-associated SNAREs (t-SNAREs | syntaxin).
The v-SNAREs are located on the vesicle membrane and the t-SNAREs are located on the target membrane. When a vesicle approaches a target membrane, the v-SNAREs and t-SNAREs come into close proximity. The v-SNAREs and t-SNAREs then form a complex, bringing the two membranes into close proximity.
This complex is held together by hydrophobic and electrostatic interactions between the SNARE proteins. Once the membranes are close enough, the SNARE complex drives the fusion of the two membranes, allowing the contents of the vesicle to be released into the target compartment.
The SNARE complex is regulated by a number of factors, including the presence of certain signaling molecules, the phosphorylation state of the SNARE proteins, and the presence of accessory proteins such as syntaxins and synaptobrevins.

Question 34

What powers mitochondrial ATP synthase, and where does this power come from?

Answer 34

ATP synthase is powered by a chemical and electrical gradient of protons across the mitochondrial membrane.
This gradient is set up by the ETC. in the ETC, NADH and FADH2 donate electrons to complexes in the mitochondrial matrix. [NADH donates its electron to Complex 1, FADH2 donates to complex 2]. The electrons will be transferred from complex 1 & 2, CoQ, Complex III Cytochrome, to complex IV.
Upon electron absorption, the complexes pump out protons into the intermembrane space. This process maintains a concentration gradient of protons across the mitochondrial membrane that favors the movement of protons back onto the mitochondrial matrix. The protons enter through ATP synthase which couples the movement of protons with the synthesis of ATP molecules.

Question 35

Name three membrane-less organelles and explain, in general, how membrane-less organelles form in cells.

Answer 35

Examples of membrane-less organelles include: nucleoli, stress granules, P bodies, Cajal bodies, pericentriolar material, and germ granules.

Intrinsically disordered regions (IDRs), protein domains that lack a distinct structural conformation, provide distinct microenvironments and solvent properties within these organelles that can concentrate and process very specific substrates, such as the different classes of RNA.
IDRs contain low-complexity (glycine, serine, and arginine-rich) sequences and repetitive motifs; they tend to phase-separate in vitro. They are tuned by interactions with RNA as well as phosphorylation or methylation.

Question 36

Explain how phase separation occurs despite the entropic costs associated with de-mixing.

Answer 36

Phase separation occurs when the free energy of the system can be lowered by separating the components into distincts phases. This can occur if the interactions between the components are strongly attractive, leading to the formation of a more stable stare when the components are separated.
The entropic costs associated with de-mixing can be overcome by the reduction in free energy resulting from the stronger attractive interactions between the separated components.
Intrinsically disordered protein-protein binding alone provides energetically favorable thermodynamics for condensate formation. The reduction in free energy achieved through increased binding at high condensate concentrations can overcome the entropic cost of de-mixing.

Question 37

Describe the two major degradative pathways in the cell. Pt1

Answer 37

The Ubiquitin-proteasome system (UPS)

The ubiquitin proteasome system is a degradation pathway that plays a critical role in the regulation of protein degradation in cells. It involves the attachment of ubiquitin, a small protein, to specific lysine residues on target proteins followed by the degradation of the ubiquitinated proteins by the proteasome, a large multi-subunit enzyme.
The E1 enzyme activates and transfers ubiquitin to the E2 enzyme, which in turn transfers ubiquitin to specific lysine residues on the target protein. The E3 enzyme helps to direct the ubiquitination of specific proteins and can also modulate the extent of ubiquitination.
Once a protein is ubiquitinated, it is recognized and targeted for degradation by the proteasome, which is composed of two subunits: the 20S proteasome, which is the catalytic core of the enzyme, and the 19S regulatory particle, which helps to recruit and unfold ubiquitinated proteins for degradation. The 20S proteasome cleaves the ubiquitinated proteins into small peptides, which are then further degraded into amino acids that can be recycled for new protein synthesis.

Question 38

Describe the two major degradative pathways in the cell. Pt2

Answer 38

The Lysosomal proteolysis pathway

Regulates extracellular proteins and cell-surface receptors.
Extracellular proteins must be internalized and trafficked to the lysosome through receptor-mediated endocytosis (RME | an acidic pH initiates the release of ligands from their receptors.), pinocytosis, or phagocytosis. Once proteins are inside the cell, they are introduced to the lysosome through vesicle fusion and the formation of a multivesicular body, activating the lysosomal proteolysis pathway.
Lysosomes are acidic, membrane-bound cytoplasmic organelles that harbor pH-sensitive hydrolases including lipases, phosphatases, glycosidases, peptidases, and nucleosidases. These enzymes play a key role in a variety of core catabolic processes that degrade macromolecules and organelles.

Question 39

How and when are proteins targeted to the proteasome for degradation.

Answer 39

Proteins are targeted to the proteasome for degradation through ubiquitination. This attachment is mediated by a set of enzymes called E1, E2, and E3, which activate, transfer, and direct the ubiquitination of specific proteins.
Proteins can be targeted for degradation by the proteasome at any time during their life cycle, depending on the needs of the cell. For example, proteins that are damaged or misfolded may be targeted for degradation to prevent them from accumulating and potentially causing harm to the cell. Proteins that are no longer needed or have fulfilled their function may also be targeted for degradation to allow the cell to recycle their constituent amino acids for new protein synthesis.
Another way in which a protein is targeted to the proteasome for degradation is through the phosphorylation of a protein kinase, which is an enzyme that adds phosphate groups to other proteins.

Question 40

Describe the structure of the proteasome and explain how each part contributes to the selective degradation of proteins.

Answer 40

The proteasome is a large multi-subunit enzyme that plays a critical role in the selective degradation of proteins in cells. It is composed of 2 subunits: the 20S proteasome (core), which is the catalytic core of the enzyme, and the 19S regulatory particle (cap), which helps to recruit and unfold ubiquitinated proteins for degradation. (Note it has 2 19S subunits on both sides of the 20S).
The 20S proteasome is a cylindrical structure that is composed of four stacked rings, two alpha subunits and two beta subunits. The alpha rings contain the active sites of the enzyme while the beta rings help to stabilize the structure of the 20S proteasome and also contain sites for regulatory proteins.
The 19S regulatory particle is composed of multiple subunits and is located at the ends of the 20S proteasome. It contains ATPase subunits that help to unfold ubiquitinated proteins and bring them into close proximity with the active sites of the 20S proteasome. It also contains specific binding sites for ubiquitin, which helps to direct the degradation of specific proteins.

Question 41

How and when do autophagosomes form?

Answer 41

formed during the process of autophagy, which is a cellular degradation pathway that involves the recycling of intracellular components. They can form in response to various stimuli, including nutrient deprivation, stress, and the presence of damaged or unnecessary proteins.
Potential membrane sources for autophagosomes include the plasma membrane, the Golgi apparatus, the endoplasmic reticulum, and the mitochondria.

Autophagosome formation begins when a group of proteins called autophagy-related proteins (ATGs) are activated and begin to form a phagophore - a small, flat membrane structure that will eventually become the autophagosome. The phagophore expands and envelops the intracellular components that are targeted for degradation, such as damaged proteins to organelles. The phagophore then fuses with a lysosome forming an autophagosome. The lysosomal hydrolytic enzymes break down the contents of the autophagosomes and the molecules can then be recycled for new protein synthesis.

Question 42

Describe the endocytic pathway from the cell membrane to the lysosome.

Answer 42

The endocytic pathway is the process by which cells take up extracellular molecules by forming vesicles around them. There are several types of endocytosis, including clathrin-mediated endocytosis, caveolin-mediated endocytosis, and phagocytosis.

In clathrin-mediated endocytosis, the extracellular molecule is first coated with a protein called clathrin, which aids the formation of a small vesicle around the molecule. The vesicle is then pinched off from the plasma membrane to become a clathrin-coated vesicle. IT then travels to an early endosome which is acidic. This low pH aids the release of extracellular molecules from the vesicle.

The early endosome then matures into a late endosome rich in lysosomal hydrolases. This eventually fuses with a lysosome.
The caveolin-mediated endocytosis follows a similar pathway with a different protein, caveolin.

Question 43

What are the three major classes of cell surface receptors? Succinctly describe how each class initiates intracellular signaling upon binding to an extracellular ligand.

Answer 43

The 3 major classes of cell surface receptors are ion channel receptors, GPCRs, and enzyme-linked receptors.

Ion channel receptors: they are activated by stimuli like changes in concentration of ions, changes in membrane potential, or binding of specific ligands. Once activated they allow for flow of ions through the plasma membrane. These ions can directly bind to an intracellular receptor and initiate signaling.

G protein-coupled receptors (GPCRs): are transmembrane proteins that are activated by the binding of specific ligands, such as hormones or neurotransmitters. These receptors are coupled to G proteins, which are intracellular signaling proteins that help to transmit the signal from the receptor to the inside of the cell.

Enzyme-linked receptors: are transmembrane proteins that are activated by the binding of specific ligands and are linked to intracellular enzymes. These enzymes can be activated or inhibited by the receptor, leading to changes in the cell’s metabolism or gene expression.

Question 44

How do signal transduction pathways selectively amplify specific signals? Describe two general mechanisms.

Answer 44

Signal transduction pathways selectively amplify specific signals through the use of receptor tyrosine kinases (RTKs) which possess a single membrane-spanning region, and related multisubunit receptors.

2 general mechanism of signal transduction: generation of second messengers, and receptor phosphorylation. Most signaling involves receptor activation of a GTP-binding protein (G protein). The activated G proteins interact with enzymes that produce second messenger molecules.

Second messengers: may arise by 2 different mechanisms.
1. Receptor mediated activation of an enzyme that catalyzes the production of the second messenger.
2. Opening or closing of ion channels in the plasma membrane.
Examples of second messengers include G proteins, Cyclic AMP, Cyclic GMP, Ca2+.

Receptor phosphorylation: a family of receptors exists that function by directly phosphorylating target proteins. Binding to these receptors activates a tyrosine kinase activity located within a cytoplasmic domain.
- This kinase activity may result in autophosphorylation of the receptor and/or phosphorylation of intracellular target proteins.
-nActivated receptor tyrosine kinase (RTK) transmit information by two mechanism:
Protein phosphorylation.
Protein-protein binding.

Question 45

What are cyclins and how do they control cell progression?

Answer 45

Cyclins are a family of proteins that controls the progression of a cell through the cell cycle by activating cyclin-dependent kinase (CDK) enzymes. They interact with a group of proteins called cyclin-dependent kinases (CDKs) to control the progression of cells through the cell cycle.

Question 46

What is Cdk? In brief, how was it discovered?

Answer 46

Cyclin-dependent kinases (CDKs) (protein kinases) are a group of enzymes that play a critical role in the regulation of the cell cycle. They function by phosphorylation specific proteins, which can alter their activity and regulate their function. CDKs are activated by binding to a specific type of cyclin protein, which helps to control the timing of theri activity during the cell cycle.
During observation of baker yeast, Hartwell observed that mutated cells stopped in the cell cycle when they were cultured at an elevated temperature. Tim Hunt observed in sea urchins that a specific protein increased in amount before cell division but disappeared when the cells divided.

Question 47

How do cells put the brakes on cell cycle progression? Describe two mechanisms.

Answer 47

Cells regulate proliferation through distinct mechanisms of growth arrest such as quiescence and senescence.
Critical to cell cycle entry is the phosphorylation of the pocket protein family - including p107, p130 and retinoblastoma protein (Rb).
One mechanism is through checkpoint control: during the cell cycle there are several points at which cells pause to ensure that certain conditions are met before proceeding to the next phase. Checkpoint proteins are activated when they detect problems such as DNA damage or improper DNA replication. They can inhibit cell cycle progression by preventing the activation of cyclin-dependent kinases.

Protein degradation - of M-cyclin by promoting addition of ubiquitin tag via APC/C.

Question 48

What is the difference between apoptosis and necrosis?

Answer 48

Apoptosis is the active, programmed process of autonomous cellular dismantling that avoids eliciting inflammation. It is a highly regulated process that is controlled by specific signaling pathways and involves the activation of caspases. It is important for maintaining the normal turnover of cells in the body and preventing the development of tumors.

Necrosis is the passive, accidental cell death resulting from environmental perturbations with uncontrolled release of inflammatory cellular contents. It is not a regulated process and typically occurs as a result of physical trauma, infection, or other forms of cellular stress. It is characterized by the breakdown of the cell membrane and the release of intracellular contents into the surrounding tissue. This can cause inflammation and tissue damage, and it is generally considered to be a pathological process.

Question 49

What are caspases and how do they contribute to apoptosis?

Answer 49

Apoptosis is mediated by caspases, which trigger cell death by cleaving specific proteins in the cytoplasm and nucleus.

Caspases are activated in response to specific signals that initiate the apoptotic process. Once activated, they cleave and destroy proteins that are necessary for cell survival, causing the cell to die. Caspases are activated by a cascade of events that begins with the activation of specific receptors on the cell surface. These receptors can be activated by various stimuli, such as DNA damage or signaling pathways that are activated in response to cellular stress.

Question 50

How do cells keep the brakes on apoptosis? Describe two mechanisms.

Answer 50

Cells can use the inhibitor of apoptosis (IAP) family.
They inhibit apoptosis by binding to some procaspase to prevent their activation.

Bcl-2 family proteins
They inhibit apoptosis by blocking the release of cytochrome c from the mitochondria.

Question 51

How do bacteria ensure equal distribution of low copy number versus high copy number components during cell division?

Answer 51

Partitioning of high copy number components such as DNA and chromosomal proteins are typically distributed by passive mechanisms that rely on the random distribution of these components during cell division.
Partitioning of low copy number components are typically distributed by an active process such as cytokinesis.
In the case of plasmids, they have partitioning mechanisms, which act resembling the mitosis process of eukaryotes and ensure proper distribution of plasmid molecules to all daughter cells.

Bacteria have various cytoskeletal homologs (i.e. homologs for tubulin and actin) that aid in partition of low copy number molecules. For example for plasmids, ParM is an actin homolog (but can do catastrophe similar to MTs) that is recruited to the outside of two plasmids and polymerizes, pushing the two plasmids away from each other towards opposite ends of the cell.

Question 52

Provide a compelling argument to support this statement: bacteria are vastly superior to eukaryotes.

Answer 52

Small size, larger numbers, wider range of feeding ability, wide range of habitats, and short generation times.

Question 53

What is GFP, and how do cell biologists use it in their experiments?

Answer 53

Green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to certain wavelengths of light. It was first isolated from the jellyfish Aequorea victoria, but has since been found in a wide variety of organisms.
Cell biologists often use GFP as a marker to track the movement and localization of protein within cells. For example, they can genetically engineer cells to express GFP-tagged proteins, which allows them to visualize the proteins in living cells using fluorescence microscopy. This can provide important information about the function and behavior of the proteins, as well as their interaction with other molecules.
GFP has been recognized as a marker in intact cells fro gene expression and protein targeting. In biological studies, it is extensively used as genetically encoded fluorescent markers.

Question 54

What is in vitro reconstitution, and why do biochemists do these sorts of experiments?

Answer 54

In vitro reconstitution refers to the process of assembling a functional biological system or pathway from purified components in a laboratory setting. Biochemists often use in vitro reconstruction as a way to study the function and regulation of individual proteins or complex molecular assemblies, such as enzymes or signaling pathways.

One reason why biochemists perform in vitro reconstitution experiments is to gain a better understanding of the mechanisms behind biological processes. By isolating and purifying individual components, researchers can test their function and interactions in a controlled environment, which can provide insights into how they work together in the context of a living cell. In vitro reconstitution can also be used to test hypotheses about the role of specific proteins or pathways in a particular process, and to identify potential drug targets.

Another reason why biochemists use in vitro reconstitution is to study the structural and biochemical properties of proteins and other biomolecules. By reconstituting these molecules in a defined system, researchers can use techniques such as X-ray crystallography, biochemical assays, and mass spectrometry to obtain detailed structural and functional information about them, This can help to stand light on the molecular mechanisms behind important biological processes, such as enzymatic reactions, and can also provide a basis for the design of new drugs or therapies.

Question 55

What are genetic screens, and why do scientists perform them?

Answer 55

Genetic screens are experiments that are designed to identify genes or genetic variations that are associated with a particular trait or phenotype. These screens are often used to study the function of individual genes, as well as to identify potential targets for therapeutic intervention.

Scientists perform genetic screens for a variety of reasons. One reason is to identify the genetic basis of a particular trait or phenotype, which can provide insights into the underlying biological processes and help to identify potential targets for therapeutic intervention. GEnetic screens can also be used to study the function of individual genes, to identify genetic variation that contribute to disease susceptibility, and to identify potential drug targets.

Question 56

Succinctly describe two different experimental approaches scientists use to identify specific genes or proteins involved in their cellular process of interest.

Answer 56

Genetic screens: involves identifying genes that are associated with a particular traitor or phenotype by analyzing the genetic makeup of individuals or populations that exhibit the trait. Scientists can use a variety of techniques to perform genetic screens, including forward genetics, reverse genetics, and chemical genetics.

Protein purification and analysis: scientists can use biochemical techniques to purify and analyze specific proteins in order to understand their function and role in a particular cellular process. This can involve isolating and purifying the protein of interest, and then using techniques such as mass spectrometry or X-ray crystallography to determine its structure and function. Scientists can also use biochemical assays to study the activity of the protein and its interactions with other molecules.

Question 57

Gibbs free energy

Answer 57

∆G = - kBT ln(C/Co)
where Co is the concentration of the reactant at equilibrium

∆Gconc. = - kBT ln(Cout/Cin)

∆Gvolt. = zFѰ
where z is the charge, F is Faraday’s constant, and Ѱ is the voltage

∆G = -nF∆E
where n is number of electrons, F is Faraday’s constant, and E is the redox potential (See Lecture 11)

Question 58

force exerted by polymerisation

Answer 58

Fmax=kBT / d * ln (C/Ccrit)

Question 59

Flux across membrane

Answer 59

J= -p (Cin- Cout)
Where p is the permeability constant

Question 60

Faraday’s constant

Answer 60

40 kBT/volts

Question 61

ln(10)

Answer 61

2

Question 62

∆G for ATP hydrolysis

Answer 62

-20 kBT

Question 63

Calculating Gibbs free energy with delta G of ATP hydrolysis

Answer 63