BIOL 0500 Midterm #3

signal transduction

• the process of converting an impulse or stimulus from one physical or chemical form to another

• cells must interpret and respond to a variety of extracellular signals received from other cells to help coordinate their behaviors (e.g., hormones, neurotransmitters, mitogens, etc.)

• always elicits a specific, coordinated cellular response/behavior, including changes in gene expression, protein expression, morphological changes, etc.

general principles of cell signaling

• communication between cells involves a signaling cell producing a signaling molecule that is then detected by a target cell

• target cells can have one of two types of receptors that recognize and respond to the signal molecule: intracellular (small and hydrophobic) and cell-surface (large and hydrophilic)

• extracellular signal molecules stimulate target cells by binding to its receptor proteins

• signal transduction begins when the receptor protein on the target cell receives an incoming extracellular signal and converts it to an intracellular signal

cell signal response speed

• cells can respond to signals quickly or slowly, depending on what needs to happen inside the cell to elicit the response

• rapid cell responses are possible when the signal affects the activity of proteins already present inside the target cell (e.g., cell movement, secretion, metabolism)

• slower cell responses occur when responses require changes in gene expression and new protein synthesis (e.g., cell differentiation, increased cell growth and division)

modes of cell communication

• there are four major modes of cell communication: endocrine, paracrine, neuronal, and contact-dependent

• many of the same types of signal molecules are used for endocrine, paracrine, and neuronal signaling

• the difference lies in the speed and selectivity with which signals are delivered to their targets

endocrine signaling

• endocrine communication is a slow-acting, long-lasting signaling system where endocrine glands secrete peptide or steroid hormones directly into the bloodstream (or plant sap) for distribution throughout the body

• cells that produce hormones are referred to as endocrine cells

• cortisol, estradiol, and testosterone are steroids; insulin is a protein; adrenaline and thyroxine are derivatives of the amino acid tyrosine

• examples: epinephrine (adrenal gland), cortisol (adrenal gland), estradiol (ovaries), insulin (beta cells of pancreas), testosterone (testis), thyroxine (thyroid gland)

paracrine signaling

• ligands are released by signal cells into the extracellular fluid through which they diffuse to induce changes in nearby cells (is a local mediator)

• cannot act over long distances since ligands can be degraded by enzymes, taken up by neighboring cells, or can only diffuse so far through the ECF

• communication between nearby cells enables rapid coordination of tissue-level responses

• examples: signal molecules regulating the inflammatory response, cell proliferation control, angiogenesis (wound healing), and autocrine signaling

autocrine signaling

• closely related to paracrine signaling

• cells secrete factors that they themselves express receptors for

• the secreted ligand binds to the receptors on the cell’s own surface, stimulating a self-response that promotes growth or survival

• during embryonic development, autocrine signaling ensures groups of identical cells differentiate into the same tissue type by allowing self-identity-reinforcement

• enables cells to create positive feedback loops, where the initial signal causes the release of more signaling molecules to rapidly amplify the signal

• cancer cells often exploit autocrine signaling

paracrine signaling examples

• epidermal growth factor (EGF) is a protein secreted by various cells to stimulate epidermal cell proliferation

• bone morphogenetic proteins (BMPs) are proteins that induce differentiation of mesenchymal stem cells into osteoblasts and cartilage

• wingless and Int-1 (WNT) are evolutionarily conserved proteins that stimulates cell proliferation (as well as embryonic development, tissue homeostasis, and cell fate)

pituitary gland

• the master regulatory gland of the body, is an endocrine gland that produces several hormones that regulate many different organs in the body

• acromegaly is a rare, slow-progressing disorder caused by excess growth hormone, leading to enlarged extremities and facial features, joint pain, a deep voice, sweating, etc.

neuronal signaling

• neuronal signals are transmitted electrically along a nerve cell axon

• once the action potential reaches the axon terminal, electrical signals are converted into chemical signals in the form of neurotransmitters, which are released into the synapse

• the neurotransmitters then diffuse across the synaptic gap to reach the target membrane

juxtacrine (contact-dependent) signaling

• type of cell-cell communication reliant upon signaling cell with membrane-bound ligand and target cell with membrane-bound receptor

• prevents accidental activation of distant cells and enables a single cell to influence a large number of immediately adjacent cells

• embryonic development: juxtacrine signaling allows adjacent cells to become more specialized to form different cell types

• Delta-Notch: a transmembrane-bound Delta protein on one cell binds directly to Notch receptors on an adjacent cell, inhibiting neighboring cells from becoming specialized in the same way as the signaling cell

Notch signaling in fruit flies

• fly nervous system originates in embryo from a sheet of unspecified epithelial cells

• isolated cells in sheet begin to specialize as neurons while neighbors remain non-neuronal and maintain the epithelium

• signals involved are juxtacrine, transmitted via direct cell-cell contacts

• lateral inhibition: cells that have begun to differentiate into neuronal cells express the membrane-bound inhibitory Delta protein, sending this juxtacrine signal to the neighboring Notch receptors to prevent mass differentiation

• lateral inhibition is necessary to ensure some cells can become support cells, maintain boundaries between tissue types, and efficiently allocate resources

Delta-Notch signaling

• when the membrane-bound signal protein Delta binds to Notch receptors on an adjacent cell, a portion of the Notch receptor is cleaved

• the cleaved portion of Notch’s cytosolic tail migrates inwards to the cell’s nucleus

• in the nucleus, the Notch tail activates Notch-responsive genes, meaning the Notch receptor itself acts as a transcription factor

• in general, Notch-responsive genes act as binary switches that control differentiation (usually via lateral inhibition) and stem cell niche maintenance

• the genes that control nerve cell production in fruit flies are Notch-responsive genes

signal-receptor axis specificity

• cells within multicellular organisms are exposed to hundreds of environmental signals

• whether or not they respond to those signals depends on whether the cells possess a receptor protein for that signal

• the extracellular signal molecule alone is not the message as the message depends entirely on how the target cell receives and interprets the signal

acetylcholine

• a neurotransmitter formed as an ester of acetic acid, an ethyl, and a triethylamine group

• demonstrates how the ligand itself doesn’t entirely convey a specific chemical message; the cell’s interpretation of the message is what constitutes the chemical message

• binding to AV node decreases heart rate

• binding to a salivary gland cell induces secretion and stimulates saliva production

• binding to a skeletal muscle cell stimulates contraction

cell surface receptors vs intracellular receptors

• two broad categories of extracellular signals: (1) large, hydrophilic, and thus impermeable (2) small, hydrophobic, and thus permeable

• hydrophilic molecules must rely on cell-surface receptors to relay their message since they are repelled by the hydrophobic interior of the phospholipid bilayer

• ligands using cell-surface receptors are far more common

• permeable molecules can diffuse across the plasma membrane and bind to intracellular enzymes or receptor proteins

• ligands that bind intracellular receptors generally regulate transcription directly, while those that bind cell-surface receptors must initiate signal transduction pathways

examples of cell-surface receptor signals

• are large, hydrophilic, or charged molecules that cannot cross the plasma membrane

• epidermal growth factor (EGF) binds to the tyrosine kinase receptor EGFR/ErbB1

• insulin, a water-soluble peptide hormone, binds to a cell-surface tyrosine kinase insulin receptor (IR)

• the neurotransmitter acetylcholine binds to nicotinic acetylcholine receptors, transmembrane ligand-gated ion channels

intracellular receptor signals

• refers to a class of cell signaling pathways that rely on receptors located inside the cell, usually the cytoplasm or nucleus

• only small, hydrophobic signal molecules can pass through the plasma membrane to use intracellular receptors

• act more slowly than cell surface receptors, typically requiring hours because their primary function is to regulate gene expression (and protein synthesis)

• steroid hormone receptors for cortisol, estrogen, and testosterone are typically located in the cytoplasm and move to the nucleus after activation

• thyroid hormone and other receptors (e.g., vitamin D or retinoic acid) are most often located in the nucleus

intracellular receptor mechanism of action

• the small, hydrophobic molecule (e.g., cortisol, estrogen) diffuses through the plasma membrane

• once inside the cell, the ligand either (1) binds to a cytoplasmic receptor that it co-translocates into the nucleus (2) binds directly to receptors in the nucleus (3) binds intracellular enzymes

• upon binding the ligand, the receptor changes shape and often dissociates from inhibitory proteins and/or dimerizes

• the release of inhibitory proteins exposes the receptor’s DNA-binding domain, allowing it to bind to regulatory regions of DNA

• dimerization activates signaling, increases ligand binding affinity, and enables binding to specific DNA sequences

• the receptor then acts as a transcription factor, either increasing or decreasing gene transcription

combinatorial signaling

• the process by which multiple signaling pathways interact to produce a more complex or finely-tuned cellular response

• allows cells to integrate multiple, simultaneous, and often opposing (positive vs negative) extracellular cues to generate unique, context-dependent responses

• positive signals initiate or amplify cellular responses (growth secretion) while negative signals act as inhibitors or terminators to prevent over-activation

• some signals are concentration dependent, others rely on specific receptors that are expressed in a highly cell type-specific manner

concentration dependent signaling molecules

• ligands that elicit different cellular responses, fates, or behaviors depending on concentration

• allows single signal molecule to trigger distinct, graded, or switch-like outcomes (e.g., low concentration causes proliferation while high concentration causes differentiation)

• morphogens often form gradients in developing tissues

cortisol signaling

• as a small, hydrophobic molecule, cortisol easily crosses the plasma membrane of target cells

• in the cytoplasm, cortisol binds to its receptor and induces a conformational change that releases inhibitory chaperone proteins and exposes a nuclear localization signal

• the activated receptor-cortisol complex then moves into the nucleus, where it binds to specific DNA sequences and acts as a transcription factor

• as a result, proteins involved in metabolism, immune system regulation, and insulin resistance are produced

extracellular receptor signaling

• a class of cell signaling pathways that rely on cell-surface receptors

• only large, polar (hydrophilic) signal molecules that cannot pass through the plasma membrane’s hydrophobic interior use extracellular receptors

• primary function is to transduce external signals into rapid intracellular signaling cascades via second messengers

• ultimately, extracellular signal cascades involve post-translational protein modifications (e.g., phosphorylation) rather than new gene transcription

• GPCRs, RTKs, and ligand-gated ion channels are the main classes of extracellular receptors

• work more rapidly tha intracellular pathways, but are generally shorter-lived and can be shut off quickly via phosphatases and GTP hydrolysis

effector proteins

• signaling molecules, often enzymes, ion channels, or transcription factors

• are primarily activated by upstream signaling molecules, most notably activated G-proteins, small GTPases like Ras or Cdc42, or lipid second messengers like PIP3 and DAG

• once the effector protein is active, it can generate or release second messengers in response to receptor activation

• examples: adenylyl cyclase (catalyzes cAMP synthesis), phospholipase C (catabolizes PIP2 into IP3 and DAG), phosphoinositide 3-kinase (activates Akt and mTOR)

general extracellular signal cascade mechanism

• a primary messenger (extracellular ligand) binds to a specific transmembrane receptor

• binding of the ligand induces a conformational change in the receptor, activating the receptor so that it can interact with or activate other intracellular proteins (second messengers and/or effectors)

• the activated receptor initiates an intracellular cascade that passes the initial message “downstream”, often involving the synthesis or release of secondary messengers (e.g., cAMP, Ca2+), or kinase activation

• the cascade ultimately activates specific effector proteins (e.g., metabolic enzymes, cytoskeletal proteins, transcription regulators) that directly influence cellular processes

• the final outcome is the cell’s response, dependent upon the receptor’s function

intracellular signaling protein functions (ARIDER)

• amplify signals received, making them stronger so that only a few intracellular signaling molecules can evoke a large intracellular response

• receive and integrate signals from multiple intracellular signaling pathways, processing them to determine the final cellular output

• distribute signals to more than one signaling pathway or effector protein that generates complex or parallel responses

• engage in feedback, with downstream signaling components influencing upstream pathway activity

• relay signals from one molecule to the next in a cascade, often via conformational changes that help the signal spread through the cell

cellular switches

• cellular switches in signaling cascades are molecular mechanisms (e.g., kinases, phosphatases, G-proteins) that turn pathways “on” or “off” by toggling intracellular proteins between active and inactive states in response to external stimuli

• once a protein is activated, it can turn on other proteins in the signaling pathway

• activated proteins exist in the active state until another switch turns them off

• kinases transfer phosphate groups to specific amino acids (most commonly serine, threonine, or tyrosine) on target proteins to activate them, often in phosphorylation cascades

• phosphatases dephosphorylate proteins, reverting them to inactive states

• G-proteins possess intrinsic GTPase activity, allowing them to switch between GTP-bound (active) and GDP-bound (inactive) states

kinases

• kinases catalyze the transfer of a phosphate group from ATP to a specific amino acid side chain on a target protein (serine/threonine kinases add to serines or threonines, tyrosine kinases add to tyrosines)

• generally act in phosphorylation cascades, where one kinase is phosphorylated and activated by upstream kinases, and then also phoshorylates the next downstream kinase

• the most common types are serine/threonine kinases (PKA, PKC, Akt, MAPK) and tyrosine kinases (RTKs)

• serine and threonine kinases are collectively termed since they share a conserved catalytic domain

• many proteins can be phosphorylated at multiple residues, a mechanism known as multisite phosphorylation

• can be regulated via negative feedback loops, scaffold proteins, and compartmentalization

multisite phosphorylation

• multisite phosphorylation involves the addition of phosphate groups to multiple amino acid residues on a single protein

• a threshhold is reached when a specific number of sites are modified, activating the protein only when the signaling kinase concentration is high enough

• the ability of an amino acid to be phosphorylated is primarily determined by its accessibility within a protein’s sequence to kinases

• turns gradual changes in kinase activity into sharp switches

adenosine triphosphate

• a purine nucleotide

• consists of a nitrogenous base (adenine), a five-carbon sugar (ribose), and a chain of three phosphate groups

• to release energy, bond between phosphate groups 2 and 3 is broken

• cleavage removes the terminal phosphate to produce adenosine disphosphate (ADP) and inorganic phosphate (Pi)

GTP-binding proteins

• intracellular signaling proteins that exist in two conformational states, depending on what is bound to the alpha subunit: GTP-bound (active) and GDP-bound (inactive)

• cell response duration is determined by how long GTP is bound to the alpha subunit

• have intrinsic GTPase activity and can shut themselves off by hydrolyzing their bound GTP to GDP

• are activated by guanine nucleotide exchange factors (GEFs) that release GDP

• are deactivated by GTPase-activating proteins (GAPs) that accelerate intrinsic hydrolysis

• two main types: trimeric and monomeric GTPases

• trimeric G proteins are composed of alpha, beta, and gamma subunits that work directly with GPCRs

• monomeric G proteins are single-subunit proteins that have a central, conserved domain that binds GDP or GTP

trimeric vs monomeric G proteins

• both have a subunit that binds and hydrolyzes GTP, acting as a molecular switch that directly regulates second messenger production

• monomeric G proteins are composed of a single subunit that resembles the alpha subunit of heterotrimeric proteins

• heterotrimeric G proteins are composed of three distinct alpha, beta, and gamma subunits

• the canonical GPCR pathway involves heterotrimeric G proteins

• monomeric are primarily involved in cell proliferation, endocytosis, transport, etc. (Ras, Rho, Rab) while trimeric are responsible for transmembrane signaling and second messenger production (cAMP

cell-surface receptor categorization

• all cell-surface receptors belong to one of three classes, differing in transduction mechanism

• ion-channel-coupled receptors: change permeability of plasma membrane to selected ions, altering membrane potential; can produce an electrical current under appropriate conditions

• G-protein-coupled receptors: activate trimeric GTP-binding proteins on the cytosolic side of the plasma membrane, which then activates or inhibits an enzyme or ion channel in the same plasma membrane, initiating an intracellular signaling cascade

• enzyme-coupled receptors either act as enzymes or associate with enzymes inside the cell, activating a wide variety of intracellular signaling pathways

barbiturates and benzodiazepines

• are depressant drugs that stimulate gamma aminobutyric (GABA) activated ion channel coupled receptors, increasing GABA activity

• GABA receptors are a type of cell surface receptor, as GABA is a polar molecule that cannot cross the lipid membrane

• relieve anxiety and have sedative effects

nicotine

• interferes with acetylcholine receptors

• stimulates acetylcholine-activated ion-channel-coupled receptors

• constricts blood vessels and elevates blood pressure

morphine and heroin

• interfere with endorphin and enkephalin receptors

• stimulate G-protein-coupled opiate receptors

• lead to analgesia (pain relief) and euphoria

curare

• interferes with acetylcholine receptors

• blocks acetylcholine-activated ion-channel-coupled receptors

• leads to blockage of neuromuscular transmission, resulting in paralysis

strychnine

• a highly toxic, intensely bitter alkaloid used primarily as a potent pesticide for rodents and coyotes

• is a CNS stimulant that blocks glycine-activated ion-channel-coupled receptors

• leads to blockage of inhibitory synapses in spinal cord and brain, resulting in severe and rigid muscle convulsions and seizures

capsaicin

• stimulates temperature sensitive ion channel coupled receptors for heat

• leads to painful, burning sensations although prolonged exposure can paradoxically lead to pain relief

menthol

• stimulates temperature-sensitive ion-channel-coupled receptors for cold

• in moderate amounts, can lead to a cool sensation; at higher doses, can cause burning pain

GPCRs

• GPCRs are the largest family of cell-surface membrane proteins

• each GPCR is a single polypeptide chain that spans the membrane 7 times

• upon binding an extracellular ligand, GPCRs undergo an activating conformational change

• the active GPCR promotes the dissociation of GDP from the alpha subunit of a G protein bound to the cytosolic side of the membrane, allowing GTP to bind

• the GTP-bound alpha subunit and beta-gamma dimer dissociate from the GPCR and each other, initiating a downstream signaling cascade

• major G-protein pathways: Gs (stimulatory), Gi (inhibitory), and Gq

guanine nucleotide exchange factors (GEFs)

• regulatory proteins that activate G proteins by promoting the exchange of GDP for GTP

• for heterotrimeric G proteins, activated GPCRs function as GEFs, releasing GDP from the G protein’s alpha subunit

• each monomeric G protein has its own associated GEF (e.g., Ras-GEF, Rho-GEF, Rab-GEF)

GTPase activating proteins (GAPs)

• inactivate G proteins by increasing the rate of GTP hydrolysis

• target both small monomeric and heterotrimeric G proteins

trimeric G protein structure

• all G proteins are made up of three subunits: alpha, beta, and gamma

• each subunit is tethered to the plasma membrane by short lipid tails

• the alpha subunit is responsible for binding guanosine nucleotides (GDP and GTP) and hydrolyzing GTP

• the alpha subunit consists of two main domains: (1) Ras-like GTPase domain that coordinates nucleotide binding and hydrolysis and (2) alpha-helical domain that covers the nucleotide-binding pocket

• the beta and gamma subunits are tightly associated and function as a single unit, anchoring the G protein complex to the membrane

G-protein mediated ion channel regulation

• mechanism by which activated GPCRs modulate ion channel activity to induce immediate changes in membrane potential

• once the GPCR activates the G protein, the G protein dissociates into its alpha subunit and gamma-beta complex

• the activated alpha subunit generally modulates cellular enzymes like adenylate cyclase, indirectly affecting ion channels through second messengers

• the activated beta-gamma complex directly binds to the intracellular faces of ion channels, increasing the permeability of specific ions

• inactivation of the alpha subunit via GTP hydrolysis returns the G protein to its inactive state

G-protein interaction w/ enzymes

• interaction of activated G proteins with membrane-bound enzymes is less rapid and more complex than G protein interactions with ion channels that lead to immediate changes in membrane potential

• G-protein and enzyme interactions are far slower because the enzymes activated by G proteins lead to the production of additional intracellular signaling molecules

• the most frequent target enzymes for G proteins are adenyl cyclase and phospholipase C

• the target enzymes can be activated by different types of G proteins, allowing cells to couple the production of small molecules to different extracellular signals

Gs proteins

• the active GTP-bound alpha subunit dissociates from the beta-gamma complex and directly activates adenylyl cyclase by binding to its catalytic domain and inducing a conformational change that increases its enzyme activity

• active adenyl cyclase removes two phosphate groups from ATP, causing the remaining AMP to form a cyclic structure, cyclic AMP (cAMP)

• cAMP then binds to the regulatory subunits of protein kinase A (PKA), forcing conformational changes that release the active kinase from a regulatory protein complex

• active PKA generally phosphorylates transcription factors or metabolic enzymes (glycogen phosphorylase that breaks down glycogen)

• PKA is a serine/threonine kinase, meaning it acts primarily by phosphorylating serine and threonine residues on target proteins

Gi proteins

• the activated GPCR acts as a GEF, inducing the alpha subunit to release GDP and bind GTP

• the alpha subunit dissociates from the beta-gamma dimer

• the active alpha subunit binds to and inhibits adenylyl cyclase, inhibiting the conversion of ATP to cAMP

• lowered cAMP leads to reduced PKA activity

• without PKA to phosphorylate target proteins, downstream signaling cascades do not proceed as usual, slowing certain cell activities

Gq proteins

• the active GTP-bound alpha subunit dissociates from the beta-gamma complex, both of which then activate phospholipase C by binding directly to the enzyme

• activated phospholipase C then cleaves PIP2 embedded in the membrane to produce two second messengers: inositol 1, 4, 5-triphosphate (IP3) and diacylglycerol (DAG)

• IP3 is a soluble molecule that diffuses through the cytosol to bind IP3-gated calcium channels on the smooth ER, inducing a rapid release of Ca2+ into the cytosol

• DAG remains in the plasma membrane and, together with the released Ca2+, activates protein kinase C (recruited from cytosol to cytosolic face of plasma membrane)

• protein kinase C (PKC) then phosphorylates its own set of downstream intracellular proteins, further propagating the signal

relative speeds of signaling pathways

• ion-channel coupled receptors are the fastest since ligand binding directly opens or closes an ion channel, leading to immediate changes in membrane potential

• GPCRs have intermediate response times because they rely on fast conformational changes to activate existing G proteins (don’t have to be synthesized)

• enzyme-linked receptors are generally the slowest since ligand binding activates enzymatic signaling pathways that influence anabolic processes like gene expression and protein synthesis

second messengers of different G protein signaling pathways

• second messengers are small, non-protein intracellular signaling molecules

• Gs: cyclic AMP (activated by adenylyl cyclase and acts upon protein kinase A)

• Gi: cyclic AMP

• Gq: IP3 and DAG (formed by phospholipase C; IP3 opens calcium channels while DAG activates PKC)

ion-channel-coupled receptors

• transmembrane receptor proteins or protein complexes that open in response to the binding of a ligand to its external face

• out of the three types of cell-surface receptors, ion-channel-coupled receptors work the fastest and simplest way

• the flow of ions across the membrane results in rapid electrical signaling

enzyme-coupled receptors

• similar to GPCRs, are transmembrane proteins that display their ligand binding domains on the extracellular side of the plasma membrane

• the cytoplasmic domain of the receptor can either act as an enzyme by itself or can form a complex with other enzymes

• discovered for their role in responses that regulate growth, proliferation, differentiation, and cell survival upon binding growth factors

• generally involved in slow (hours long) cell responses, with effects requiring intracellular transduction steps that lead to changes in gene expression

• the most common types have cytoplasmic domains that function as receptor tyrosine kinases (RTKs)

growth factors

• extracellular signals that regulate cell growth, proliferation, differentiation, and survival

• primarily bind to enzyme-linked receptors and elicit relatively slow responses

• can mediate and direct rapid reconfigurations of the cytoskeleton, controlling cell shape and movement

receptor tyrosine kinases

• a large class of enzyme-coupled receptors, abnormalities in which play a major role in cancer development

• consist of an extracellular ligand-binding domain, a single alpha helix transmembrane domain, and in intracellular cytoplasmic domain that possesses tyrosine kinase activity (it phosphorylates tyrosine residues on specific intracellular proteins)

• binding of a ligand to its extracellular domain triggers dimerization, which then activates the cytoplasmic kinase domains

• the cytoplasmic kinase domains, causing them to add phosphate groups to add phosphate groups to the tyrosine residues on the other tail

• each phosphorylated tyrosine serves as a specific binding site for different downstream intracellular signaling protein

Ras structure

• a small GTP-binding protein attached via a lipid tail to the cytosolic face of the plasma membrane

• a monomeric GTPase made up of only one unit that resembles the alpha subunit of a G protein

• adaptor proteins bound to the cytosolic tail of activated RTKs recruit Ras-GEF, which promotes GDP-GTP exchange on Ras

• similar to a G protein’s alpha subunit, Ras cycles between an active GTP-bound state and an inactive GDP-bound state

Ras-GEF

• RasGEFs: Ras guanosine nucleotide exchange factors

• are recruited from the cytoplasm to the plasma membrane once a receptor has received an extracellular signal like a growth factor

• bind to Ras proteins and induce a conformational change that allows GDP to dissociate from the protein

• due to high cytoplasmic concentrations of GTP, GTP quickly replaces the lost GDP to create the active Ras-GTP complex

Ras and cancer

• in its active state, Ras promotes activation of a serine/threonine protein kinase phosphorylation cascade

• Ras activates a mitogen activated protein kinase (MAPK) signaling module

• mitogens are extracellular signal molecules that stimulate cell proliferation

• MAPK phosphorylates various downstream signaling pathways that control gene expression, thereby regulating cell proliferation and survival

• about 30% of human cancers contain activating mutations in a Ras gene; the protein cannot shut itself off, promoting uncontrolled cell proliferation and cancer development

PI-3-kinase-AKT signaling pathway

• insulin-like growth factor-1 (IGF) is a peptide hormone that promotes cell survival and proliferation through RTKs

• IGF activates RTK, causing the receptors to dimerize and phosphorylate specific tyrosine residues on the opposite receptor's tail

• the phosphorylated RTK activates the phosphoinositide 3-kinase (PI3K), which then phosphorylates the membrane-associated inositol phospholipid

• phosphorylated inositol phospholipid recruits the serine/threonine kinase Akt from the cytosol to the plasma membrane, where it is phosphorylated by protein kinase 1 and protein kinase 2

• activated Akt can then phosphorylate the Bad protein, inactivating it to release Bcl2, an anti-apoptotic protein

• Akt also indirectly activates Tor by phosphorylating and inactivating a Tor-inhibiting protein, thereby promoting cell growth

activated Akt kinase pathways

• once activated, Akt is released from the plasma membrane and phosphorylates various downstream proteins on specific serine and threonine residues

• Bad: Akt phosphorylates and inactivates the pro-apoptotic protein Bad; phosphorylation of Bad leads to its sequestration in the cytoplasm so that it can no longer promote apoptosis

• Tor: Akt can phosphorylate and active Tor, a serine/threonine kinase that stimulates cell growth by both enhancing protein synthesis and inhibiting protein degradation; Tor signaling disruptions are associated with multiple diseases such as cancer and neurodegeneration

how do mutant proteins help determine where intracellular signaling molecules bind?

• mutant proteins can help determine exactly where an intracellular signaling molecule binds

• consider a pair of RTKs in which the tyrosines have been replaced by phenylalanine

• since the phosphorylated tyrosines on RTK’s cytoplasmic tails bind the intracellular signal proteins that propagate signals throughout the cell’s interior, the mutant receptors block the signal cascade

• to determine the exact effect on the cell’s response to the signal, you must isolate cells that have only mutant receptors

how can genetic screening help determine the function of cell signaling proteins?

• genetic screens identify pathway components by systematically mutating genes to observe how the resulting cellular signaling pathway changes

• mutations reveal which proteins are required for signaling

• order of proteins can be determined (e.g., downstream vs upstream)

• can use constitutively active proteins to test position: if Ras restores signaling, the mutated protein is upstream of Ras; if Ras does no restore signaling, the mutated protein is downstream of Ras

stages of animal embryonic development

• the first five stages are common to all eukaryotes: gametogenesis, fertilization, cleavage, gastrulation, and organogenesis

• via several rounds of mitotic divisions without growth, the embryo begins as a zygote and becomes blastomeres (2,4,8 cells), morula (16-32 cells), bilaminar disc (inner and outer cells), and a trilaminar disc (ectoderm, mesoderm, endoderm)

• many animals, particularly insects, amphibians, and marine invertebrates, undergo a larval stage once the egg has hatched

• in humans, organogenesis immediately precedes the fetal stage

gametogenesis

• the biological process of forming mature, haploid gametes from diploid primordial germ cells

• occurs via meiosis and differentiation in the gonads

• three main stages: mitotic proliferation of precursor cells, meiosis, and structural maturation

• either spermatogenesis in males or oogenesis in females

blastomere vs blastocyst vs blastula

• blastomeres are the individual, undifferentiated, totipotent cells produced by the initial cleavage of a fertilized egg

• blastula is the early hollow-sphere stage of an embryo, characteristic of many animals, consisting of a single layer of cells

• the blastocyst is the mammal-specific, hollow, and highly differentiated embryo formed after the blastomeres differentiate into the inner cell mass (embryonic) cells and outer trophoectoderm (placenta)

fertilization

• sperm are attracted to ovulated eggs by the chemical signals released by the egg and the supporting cells that surround it (progesterone)

• the sperm undergoes maturation changes in the female reproductive tract that enhance its motility

• once the sperm finds the egg, it migrates through a protective layer of cells and then binds and tunnels through the zona pellucida (egg coat)

• the egg then releases cortical granules that alter the zona pellucida so that it is impermeable to other sperm to prevent multiple fertilizations

• fusion between the sperm and egg membranes initiates metabolic reactions within the egg that trigger completion of meiosis II and the onset of embryonic development

• finally, the haploid sets of male and female chromosomes fuse to create a single diploid zygote nucleus

cleavage

• the initial stage of embryogenesis immediately following fertilization

• involves a rapid series of mitotic cell divisions (~5-6) occurring without cell growth (embryo remains roughly the same volume as the original zygote)

• produces progressively smaller cells called blastomeres that are held together through cell adhesion molecules

• cleavage begins when the zygote divides into two blastomeres

• compaction begins at the 8-cell stage, and as the 8 cells transition to the 16-cell morula, they divide either parallel (symmetric) or orthogonal (asymmetric) to the apical-basal axis

• the morula is after about three to four days (rounds) of cleavage, the embryo becomes a solid, mulberry-shaped cluster of 16 cells that forms around day 3 or 4

• around day 5 to 6, as cell division continues and the blastocoel (cavity) forms, the embryo matures into a blastocyst via blastulation

compaction process

• a key aspect of the embryonic cleavage process occurring during the transition from the 8-cell stage to the 16-cell morula

• blastomeres of preimplantation embryos begin to flatten, causing them to cluster tightly together

• cell-adhesion molecules concentrate at the interfaces between cells, increasing cell-cell contacts

• since the blastomeres cluster tightly together, compaction reduces surface area and enhances cell communication

• at the 8-cell stage, all 8 cells begin on the “outside” since the inner cavity has not yet been formed

• however, as the 8-cell embryo undergoes compaction, it also establishes apico-basal polarity as the first real sign of differentiation

morula polarization

• the 8-cell embryo undergoes compaction, where the blastomeres of the preimplanatation embryo begin to flatten against each other and differentiate based on position

• as the embryo divides to become the 16 to 32-cell morula, the blastomeres divide either parallel or orthogonal to the apical-basal axis

• the apical portion faces the external environment (zona pellucida), while the basal portion faces the interior of the embryo and is rich in adhesion molecules

• parallel (symmetric) division gives rise to two daughter cells that inherit both apical and basal proteins, remaining polar and in the outer layer of the embryo

• orthogonal (asymmetric) division, gives rise to one polar outer cell (inheriting both apical and basal components) and one apolar inner cell (inheriting only the basal components)

inner cell mass vs outer trophoblast cells

• at the 16-32 cell stage (transition from morula to blastocyst), the embryo has officially split into two distinct lineages: the inner cell mass and outer trophoblast

• inner cells are clustered on the inside of the embryo, pushed to one side of the blastocoel (fluid-filled cavity)

• inner cells are apolar since they are surrounded by other cells on all sides, while outer cells are polar since they have an outwards-facing portion

• inner cells also maintain pluripotency (and Oct4 and Nanog expression) as they are protected from external signals that may induce differentiation

• outer cells are highly polarized and begin differentiation (losing pluripotency) as the embryo becomes a blastocyst

• these outer cells become the specialized outer layer (trophoectoderm) that eventually differentiates into the placenta, while the inner cell mass forms the embryo

transcription factors involved in preimplantation embryonic development

• Oct4 is expressed in the inner cell mass, required for establishing the inner cell mass in vivo

• Cdx2 is expressed in the trophoectoderm to drive TE gene expression, maintain epithelial integrity, and facilitate the cell proliferation necessary for proper implantation

• Cdx2 also binds to the Oct4 promoter in TE cells, suppressing Oct4 expression in the TE to maintain distinction between ICM and TE cells; without it, Oct4 is ectopically expressed in the TE

• conversely, in the inner cell mass, Oct4 represses Cdx2 expression

• mutual antagonism between Oct4 and Cdx2 establishes the blastocyst

• Nanog promotes rapid cell division and entry into the S-phase, ensuring ICM cells stay undifferentiated (pluripotent)

blastulation

• the final stage of cleavage in which the blastocoel is formed, turning the morula into a blastocyst (mammalian blastula)

• the blastocyst is characterized by a hollow ball of 100-200 cells that have begun differentiation

• Na/K pumps in the outer trophoblast cells create an ion gradient that forces water inward to form a fluid-filled cavity at the center of the solid morula called the blastocoel

• once the differentiated cells rearrange into two distinct areas (the inner cell mass and outer trophoblast), the structure is known as a blastocyst

• the inner mass cells are pushed to one pole of the embryo via fluid accumulation

• the inner cell mass cells are pluripotent and properly form the embryo

implantation

• 6-12 days post-fertilization, the blastocyst breaks free from its outer shell (zona pellucida) and aligns and attaches to the uterine lining

• trophoblast cells (future placenta) differentiate and invade the stroma which allows the blastocyst to embed within the uterine endometrium to initiate pregnancy

gastrulation

• an early embryonic process occurring after cleavage and implantation, around day 14-17 of human development

• the embryo reorganizes into three primary germ layers, marking the transition from a simple cell mass to a developing fetus

• the two-layered (inner vs outer) embryo transforms into a three-layered structure (endoderm, mesoderm, and ectoderm)

• the endoderm is the innermost layer, forming the digestive and respiratory tracts

• mesoderm is the middle layer that forms the muscles, bones, blood, and connective tissues

• the ectoderm is the outermost layer that gives rise to the nervous system and skin

• marks the beginning of organogenesis and establishes the cranial/caudal axis and body symmetry

organogenesis

• the critical developmental process following gastrulation, where the three germ layers (ectoderm, mesoderm, and endoderm) differentiate and fold to form functional organs

• movement and interactions between the three layers of the gastrula generate rudimentary, precursor organs

• the ectoderm forms the nervous system and skin

• mesoderm creates muscles, skeleton, and circulatory system

• endoderm forms the digestive and respiratory linings

sexual reproduction

• biological process in which two parents combine genetic material through fusion of haploid gametes to create a genetically unique diploid zygote

• with most sexually reproducing organisms, males and females produce different types of gametes

• introduces genetic diversity by mixing parental DNA

gametes

• mature sex cells that fuse with another gamete of the opposite sex during fertilization to create a diploid (2n) zygote

• are haploid (n) cells produced through meiosis to propagate genetic information to the next generation

• primordial germ cells are the earliest precursor cells in an embryo that develop into gametes

• spermatogenesis in the testis produces sperm, while oogenesis in the ovary produces eggs

germ line cells

• the germ line refers to the lineage of reproductive cells that differentiates into gametes

• begins with primordial germ cells that originate in the early post-implantation embryo and migrate to the developing gonads

• in the gonads, PGCs ultimately give rise to sperm in males and oocytes in females via gametogenesis (spermatogenesis and oogenesis)

• mutations in germ line cells can be inherited as their function is to pass DNA to offspring

• undergo mitosis to maintain the germline and meiosis to go from diploid to genetically unique haploid gametes

• primordial germ cells are specified either by inheritance (germ plasm in C elegans) or induction (extrinsic signals)

germ line vs somatic line cells

• both originate from a single fertilized egg but diverge early in embryonic development

• ploidy: somatic cells are diploid (2n); primordial germ cells are diploid (2n) cells that give rise to precursor cells that undergo meiosis to produce functional haploid (n) gametes

• function: germ line cells produce gametes and pass DNA to offspring, while somatic cells build and maintain the body

• developmental fate: germ line cells are restricted to the reproductive lineage, while somatic cells differentiate into many other specialized cell types

• division: germ cells can undergo both mitosis and meiosis, while somatic line cells can only undergo mitosis; once a germ cell enters meiosis, it loses the ability to divide by mitosis

• mutations: somatic cell mutations die with the individual, while germ cell mutations are inherited by the next generation

germ cells vs gametes

• germ cells are the diploid precursor cells present in the gonads that give rise to gametes (spermatogonia and oogonia)

• germ cells are the diploid (2n) cells that undergo mitosis and meiosis to produce haploid daughter cells (n)

• the haploid daughter cells then undergo gametogenesis to become ova and spermatozoa

germ plasm specification of germline cells

• is one hypothesis for germ line specification (the process by which certain cells are designated to become PGCs) in which germ cells are established via germ plasma inheritance

• somatic cells have factors that push them toward non-germ cell fates (e.g., skin, muscle, neuronal, etc.)

• the germ plasm is a specialized region of the cytoplasm found in the early embryo, containing large granules rich in RNA and proteins that serve as the maternal blueprint for germ cell fate

• germ plasm-carrying cells often exhibit a temporary block in transcription during early embryogenesis, suppressing somatic differentiation signals

• as the original germ cell divides, each daughter cell inherits the inhibitory machinery that keeps them from losing their germ cell identity

• primordial germ cells continue to be produced via mitosis of parent germ cell

• in contrast to the induction hypothesis, the germ plasm specifies these cells immediately

induction-based specification of germline cells

• mechanism by which primordial germ cells (PGCs) are specified in early embryos via paracrine signaling between cells

• bone morphogenic protein (BMP) signaling in the posterior embryo induces PGCs

• BMP4 and BMP2 are expressed in the extraembryonic ectoderm to induce early embryonic cells in the epiblast to form primordial germ cells

• BMPs bind to a receptor complex, activating the receptor to cause phosphorylation of SMAD1 or SMAD5

• phosphorylated SMAD1/5 form a complex with SMAD4, which translocates to the nucleus to activate transcription factors for PGC specification

• once specified, PGCs migrate from the yolk sac to the gonadal ridge, where they transition into either oogonia or spermatagonia to become gametes

GFP imaging

• green fluorescent proteins (GFPs) are biological tools used to visualize proteins, cells, and physiological processes in real-time

• can be used to visualize and isolate living germ cell lines, particularly during embryogenesis and spermatogenesis

BMPs during embryonic development

• bone morphogenetic protein signaling in the posterior of the embryo induces formation of primordial germ cells

• TE cells secrete BMP dimers that diffuse through the extracellular matrix, binding to complexes consisting of type I and type II serine/threonine kinase receptors in the posterior embryo

• the activated type II receptor phosphorylates the type I receptor

• the type I receptor then phosphorylates Smad 1/5/8, which then forms a complex with Smad 4

• the Smad 1/5/8-Smad4 complex moves into the nucleus to activate key transcriptional regulators of germ cell fate

mitosis vs meiosis

• mitosis starts with a 2n cell, which duplicates its chromatids during S phase to allow the cell to divide back into two identical 2n daughter cells

• meiosis I produces two haploid cells, and meiosis II further separates the chromatids to produce four genetically diverse haploid cells

• gametes are the haploid cells generated from diploid cells via reductive cell division (meiosis)

• meiosis generates 4 nonidentical nuclei, while mitosis produces two identical diploid nuclei

• duplicated homologous chromosomes pair up during metaphase I of meiosis, while individual chromosomes line up during mitosis

meiosis I

• before meiosis begins, the cell replicates its DNA so that each chromosome consists of two identical sister chromatids (2n)

• prophase I: the first 5-step phase of meiosis I in which chromosomes condense, homologous chromosomes pair up, and crossing over occurs to create new gene combinations

• metaphase I: homologous chromosomes align along the center of the cell, with maternal and paternal chromosomes being assigned randomly on either side of the metaphase plate

• anaphase I: homologous chromosomes are pulled to opposite poles while sister chromatids remain together

• telophase I and cytokinesis: the cell divides into two haploid daughter cells, each with 23 chromosomes (n)

meiosis II

• the second division phase of meiosis that closely mirrors mitosis

• prophase II: chromosomes condense again if they decondensed in telophase I and the nuclear envelope breaks down; centrosomes duplicate and move to opposite poles to form a new spindle apparatus

• metaphase II: chromosomes consisting of two sister chromatids line up individually along the metaphase plate; kinetochore microtubules attach to each from opposite poles

• anaphase II: centromeres holding the sister chromatids separate, and the now individual chromosomes are pulled toward opposite poles by the spindle fibers

• telophase II: nuclear envelopes reform around the sets of chromosomes at each pole, and the chromosomes begin to decondense

• cytokinesis: the cytoplasm divides, producing a total of four genetically unique haploid (n) daughter cells

phases of prophase i (LZPDD)

• prophase I of meiosis is the longest and most complex stage since it includes the processes essential for introducing genetic diversity and proper chromosome segregation

• leptotene: prophase begins, duplicated chromosomes (made up of two sister chromatids) start to condense

• zygotene: homologous chromosomes begin to pair up by formation of the synaptonemal complex, meaning the chromosomes are in their bivalent (associated) form

• pachytene: homologs are fully synapsed (precisely paired) and crossing over (homologous recombination) occurs between non-sister chromatids existing on different chromosomes; points of contact at cross-over sites are called chiasma

• diplotene: synapsis ends with the disassembly of the synaptonemal complex; chromosomes begin to separate but remain connected at the chiasmata

• diakinesis: prophase ends with the nuclear membrane disintegrating, the chiasma becoming more evident, and the homologous chromosomes moving farther away

chromosome recombination

• homologous recombination occurs during prophase I of meiosis

• during pachytene, recombination enzymes create double-strand breaks (DSBs) in either the maternal or paternal chromatid, with non-sister chromatids physically breaking at the same location

• nucleases digest the ends of strands, leading to the formation of overhangs

• using its overhang, one broken DNA strand will invade the matching sequence on its homologous non-sister chromatid and base pairing occurs between maternal and paternal DNA as each chromatid uses its homolog as a template for repair

• the DNA is copied and ligated by DNA ligase, forming a structure with two crossovers (double stranded DNA=each fragment has two exposed 3’ overhangs)

• the physical crossover points are called chiasmata, which look like X-shaped connections between homologous chromosomes

• once meiosis I finishes, the chromatids separate out from each other and each contains a segment of maternal DNA and a segment of paternal DNA

synaptonemal complex

• the synaptonemal complex is a protein structure that holds homologous chromosomes together during prophase I, facilitating the process of crossing over

• cohesins hold sister chromatids together and are attached to axial core proteins

• axial core proteins form a central scaffold along the length of a single chromosome, organizing each homolog into a linear structure and serving as attachment sites for the synaptonemal complex via transverse filaments

• the axial cores of homologous chromsomes align about 100nm apart, forming lateral elements of the synaptonemal complex (looks like a ladder with transverse filaments as rungs and axial cores as sides)

• transverse filaments connect the lateral elements of each homolog, forming a zipper like structure that allows the inner arms of chromosomes to interact with one another (enabling recombination machinery to carry out crossing-over)

metaphase I of meiosis I

• second stage of meiosis I where homologous chromosome pairs (tetrads) align randomly along the cell’s metaphase plate to allow for over 2 to the 23rd possible genetic combinations in a single gamete

• chiasmata (crossover sites) hold maternal and paternal homologs together, ensuring they stay paired as a tetrad

• in metaphase I, cohesins are found along the length of both chromosome arms to keep homologs together as a bivalent after recombination

• cohesins are also found at the centromere, joining sister chromatids

• although each chromatid has its own kinetochore, kinetochores found on sister chromatids in a single homolog function as a single unit; microtubules attach to both sister chromatids from the same pole to pull the entire homolog to one side of the cell

• the homologous chromosome (other member of the pair) is attached to microtubules from the opposite pole

anaphase I of meiosis I

• the cohesins that held together the arms of homologous chromosomes are degraded, allowing the homologs to separate

• the depolymerization of spindle fibers creates pushing and pulling forces that move homologs to opposite poles of the cell

• the cohesins found at the centromeres are not degraded to ensure sister chromatids remain together through meiosis II

how does meiosis introduce genetic diversity

• during crossing over in prophase I of meiosis I, corresponding segments of non-sister chromatids (one maternal and one paternal) in a homologous pair are swapped

• the swapped segments are equivalent in terms of the gene locus but often contain different alleles, creating recombinant chromosomes with unique allele combinations

• independent assortment refers to the random pairing of homologous chromosomes at the metaphse plate during metaphase I

• independent assortment means that different combinations of maternal and paternal chromosomes are distributed into gametes

• since humans have 23 chromosomes and each chromosome can be distributed to either side of the cell, independent assortment alone introduces 2^23 possible chromosome combinations for a single gamete

errors during meiosis

• nondisjunction in meiosis I: failure of homologs to separate, resulting in two gametes with an extra chromosome (n+1), and two gametes with a missing chromosome (n-1)

• nondisjunction in meiosis II: failure of sister chromatids to separate, producing one gamete with an extra chromosome, one with a missing chromosome, and two normal

• structural rearrangements: deletions, duplications, translocations, inversions, etc.

• unequal crossing over: if homologs do not align properly during prophase I, similar or repetitive sequences on the DNA can pair with each other so that whne the chromosomes break and recombine, one chromosome experiences deletion while the other gets a duplication

trisomy 21

• more commonly known as Down syndrome, is a genetic condition caused by an extra copy of chromosome 21 (2n+1)

• can occur in some or all somatic cells

• usually results from nondisjunction during maternal meiosis I, where homologous chromosomes fail to separate

• is predominantly due to issues in meiosis I since it involves problems with recombination and the prolonged arrest of oocytes, which leaves homologous chromosomes more vulnerable to separation errors during the first division

• age is the main risk factor for Down syndrome because, as women age, the cohesins that hold chromosomes together degrade and it increases the risk of premature separation of chromosomes

mutations

• mutations refer to a permanent change in a given DNA sequence

• can be silent (no effect on gene function), deleterious (disrupt gene function), or advantageous (enhance gene function)

• may lead to genetic diversity, cancer in somatic cells, or birth defects in germ cells

• can be caused by mistakes in DNA replication or DNA damage

alleles

• alleles are one of two or more alternative forms of a gene (e.g., B for brown eyes and b for blue eyes)

• alleles arise via mutations and are found at the same locus (position) on homologous chromosomes

• homozygous: two identical alleles for a gene (BB or Bb)

• heterozygous: two different alleles for a gene (Bb)

• hemizygous: possessing only one allele instead of two (genes on X chromosome in males)

genotype

• refers to an organism’s unique genetic makeup, specifically the combination of alleles they have inherited from their parents

• an organism’s genotype acts as the internal instructions behind their observable characteristics (phenotype)

• examples: BB, Bb, bb

phenotype

• refers to the set of observable characteristics of an individual resulting from the interaction of its genotype with the environment

• not limited to visible traits like hair color, but include molecular and metabolic characteristics like blood cell counts or enzyme levels

wild type

• wild type refers to the most common, “normal” allelic form of a gene or trait in a population

• considered the standard/reference

• example: if most flies have red eyes, then red becomes the wild type