basically everything lol

define signalling

the cascade of processes by which an extracellular stimulus (typically neurotransmitter or hormone) effects a change in cell function

what is a cascade

cascade is a central consent which refers to a chain reaction where one event triggers another eventually leading to a functional change in the cell

what is neurotransmission

communication between neurons allows for cognitive processes (thinking) (glutamate is an example of a primary excitatory neurotrasmitter in the brain)

types of cell signalling

1) signalling through an ion channel (open to allow ion flow)

2) signalling through receptor proteins (usually on the cell surface respond to stimuli to initiate an internal cascade)

different types of ion channels

K+ channels on membrane - allow potassium to leave the cell

Na+ channels - allows sodium to enter the cell

Ca2+ channels - allow calcium to enter the cell

(in vitro fertilisation sperm triggers calcium oscillations - repetitive and transient increases in concentrations and these oscillations are both necessary and sufficient to trigger embryo development)

what is calciums versatility defines by

1. breadth - it controls almost everything, from metabolic enzymes to gene transcription

2. speed - it operated on timescales from microseconds (nerve firing) to days (developmental processes)

how can ions cross the lipid bilayer

because ions carry charges they cannot cross the lipid bilayer without assistance

carrier proteins - undergo large conformational changes to move ions

ion channel - have much smaller conformational changes, essentially acting as an aq. pore

what is the concentration of sodium potassium and calcium ions inside vs outside

Na + - 5-15 vs 145 

K+ - 140 vs 5

Ca2+ - 10^-4 vs 1-2 (steepest gradient which creates a massive gradient that cell tap into for signalling)

different ion levels in organelles

ER has high calcium compared to the vanishingly low nanomolar levels in the cytoplasm

lysosomes act as the recycling centre and are full of acid (low pH) compared to the neutral cytoplasm

mitochondria maintain a proton gradient to drive ATP synthesis

what is passive transport

follows the concentration gradient “downhill” and no energy is required

what is active transport

moves ions “uphill” against the gradient and required energy

what does flow of ions depend upon

flow of ions depends on the chemical (concentration) and the electrical (membrane potential) 

most cells are slightly negative on the inside which attracts positive ions (facilitating flow) 

if the potential flips to positive flow is hampered

what does sodium potassium pump do

the sodium potassium pump generates the opposite gradients for sodium and potassium simultaneously

the specific ratio of ions moves - 3Na+ out and 2K+ in - per ATP molecule

6 step cycle in sodium potassium pump

1. protein binds Na+ inside

2. ATP transfers a phosphate to the protein

3. this induces a conformational change that tightens (occludes) the binding site so sodium can  no longer bind and is released outside

4. the change exposes a potassium binding site and K+ binds

5. the phosphate group falls off reversing the change and releasing K+ inside

what are the 2 Ca2+ pumps

PMCA: a P-type ATPase that removes calcium from the cell to maintain the gradient across the plasma membrane

SERCA: moves calcium from the cytosol into the ER

how does secondary transport work

secondary transport does not use ATP directly, instead it uses the potential energy stored in an existing gradient (usually sodium) to move a different ion against its own gradient

NCX (transmembrane portien)

• an antiporter (exchanger)

• 3 Na+ in to drive 1 Ca2+ out

• it hijacks the energy of sodium moving down its gradient into the cell to force calcium out of the cell against its gradient

what do voltage-gated ion channels respond to and their structures

voltage-gated ion channels - respond to changes in membrane potential

all voltage gates channels share a conserved structure with helices S1-S6

S1-S4 form the voltage-sensing domain

S5-S6 form the pore domain where ions actually flow

what do ligand gated channels respond to

respond to small molecules binding on either side of the membrane

what do mechanically gates channels respond to

respond to physical force or membrane tension (eg in stretching blood vessels)

structure of K+ channel

(tetramer of separate units) 

in sodium and calcium channels these four building blocks are joined into a single polypeptide likely due to gene duplication during evolution

how come potassium ions can exclude sodium ions even though sodium is smaller

MacKinnon’s atomic structure revealed that the selectivity filter is the hole that select for K+

the filter contains oxygen atoms arranged so that a dehydrated potassium ion fits snugly and is stabilised by 4 oxygens

sodium is too small to be stabilised by all 4 oxygens (only fits 2) making the passage thermodynamically unfavourable

the S4 helix contains positive charges (triplets of Arginine R and lysine K)at regular intervals which are normally neutralised by other residues but they are the specific part of the protein that sense potential changes 

at rest S4 is in a “down” position 

when the membrane depolarises the change in potential causes a clash of charges and the S4 helix moves up

this movement is physically coupled to the opening of the channel’s pore

what is electrical signalling

the use of membrane potential changes to transmit information

what is membrane potential

voltage difference between the interior and exterior of the cell (resting value is typically -70mV)

the membrane potential can sway ionic flux

if there were no potential flow would be reduced because there would be no negative charge to attract positive ions into the cell

conversely flipping the potential to positive (depolarisation) hampers the flow of ions

what does the membrane potential arise from

the potential arises from high Na+ outside and high K+ inside

although cells are packed with ions they are generally electrically neutral overall 

any small mismatch is mopped up by fixed anions within the cell

what are fixed anions

internal molecules that mop up excess positive charge to maintain near-neutrality

what is the equilibrium potential

the point where the permeable ion is subject to equal and opposite chemical and electrochemical gradients

what is the Nerst equation

Nernst equation: V = (RT/zF)ln(C_o/C_i) (this equation calculates the equilibrium potential for a single ion)

where:

R = gas constant

T = absolute temperature in K (C^o + 273.15)

F = Faraday's constant

z = valence (the charge of the ion, eg +1 for K+)

for body temp (37C) the constant part of the equation is 0.027

what is resting potential due

arises primarily due to K+ and leak channels

what are leak channels

 channels that allow K+ to leak out and be drawn back setting the resting potential 

leak channels make the membranes more permeable to K+ than anything else (therefore the resting potential stays very close to K+ equilibrium potential of -90mV)

what is an action potential

the action potential is a propagating wave of change in membrane potential

very fast

races down the axon (which can be over 1m long) to the terminal branches

what is an axon

longest projection of a neuron that carries the action potential

what is a synapse

the junction where the electrical signal “jumps” to another cell

what are action potentials due to

action potentials arise due to Na+ and K+ channels

it starts when a stimulus causes the cell to depolarise

Na+ channels sense the depolarization with their positively charged amino acids and open

sodium flood in bringing positive charge

however they are designed to inactivate quickly to prevent catastrophic loss of homeostasis

in an active depolarising neuron Na+ permeability is higher than K+ (spike on potential graph at +50mV as membrane potential of sodium is +60mV)

explain depolarisation

K+ channels activate after a delay and are not as fast as Na+ channels

the delay allows the membrane to depolarise first 

when they eventually open they pull the potential back to rest (K+ equilibrium) resetting the system

what is needed to get the signal across the synapse

to get the signal across the synapse a voltage-gated calcium channel is needed

these channels are at the synaptic membrane 

when the action potential arrives they open calcium floods in and triggers exocytosis releasing little packets of neurotransmitter into the synaptic cleft

what are quanta

 little packets of transmitter (identified by NW (nobel winner) Bernard Katz)

what types can a signal be

ligand-gated ion channels receive the signal

signalling can be excitatory (stimulating the next neuron) or inhibitory (dampening the signal)

the brain integrates these “on and off” signals in real-time as a form of computation 

why do inotropic glutamate receptors differ from voltage-gated structures

they have an inverted pore topology

looks like the S5/S6 helices but flipped

what is inotropic

channels that allow ions through directly

what type of transmitter is glutamate

glutamate is the major excitatory transmitter

these channels let in positive ions (Na+ and Ca2+) causing a pulse of depolarisation that continues the signal

what are GABAa receptors

• pentametric structure (5 subunits)

• are cys-loop receptors due to a conserved disulphide bond 

• conduct chloride (negative) - influx of negative charge makes the neuron less likely to fire hence they are inhibitory

what are nicotinic acetyl choline receptors (nAChR)

• like GABA these are cys-loop pentamers

• conduct sodium making them excitatory

how do nerves communicate with muscles

neuromuscular junction NMJ

what is the process of activated NMJ

process of activated NMJ is similar to nerve-nerve

action potential → Ca2+ influx → ACh release

ACh binds to nAChRs on the muscle and sodium floods in and the muscle depolarises

depolarisation initiates excitation-contraction (E-C) coupling

the depolarisation of the muscle membrane triggers calcium release from intracellular stores (the sarcoplasmic reticulum SR) which then engages the cytoskeleton to cause contraction

what is a receptor protein

the key primary target for signalling molecules at the top of a signalling cascade

what is a signalling cascase

the sequential process of events triggered by receptor binding

eg extracellular signal molecule binds to receptor protein triggering a cascade of intracellular signalling proteins which act on target proteins (eg metabolic enzymes, gene regulatory proteins, cytoskeletal proteins) to alter cell function

where are most receptor proteins

most receptor proteins are on the cell surface 

these bind hydrophilic stimuli as those molecules are confined to the outside of the cell

conversely intracellular receptors require hydrophobic stimuli that can pass through the lipid bilayer to reach them

what are the 2 molecular switches - systems that can be toggled “on” and “off”

1. phosphorylation

phosphate is transferred from ATP onto a protein to activate it

signal turns off when the phosphate falls off

2. GTP Binding (G proteins)

in the inactive state (off) the protein is bound to GDP (disphosphate)

in the active state (on) it is bound to GTP (triphosphate)

what are G-proteins

proteins that act as molecular switches using GTP/GDP binding

what are second messengers

small intermediary molecules that broadcast a signal within the cytoplasm

act early on in the signalling cascade

when a receptor binds its stimulus it often triggers the production of these molecules to do the work inside the cell 

cyclic AMP (cAMP), cyclic GMP (cGMP), diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3)

what are the types of receptor proteins

classified into 2 groups: enzyme-linked receptors and G-protein-couple receptors (GPCRs)

GPCRs use the GTP switching mechanism while enzyme-linked receptors act as enzymes themselves

GPCRs

GPCRs are a huge family of proteins

by scouring the human genome scientists have found roughly 800 different GPCRs

are clinically vital 

many common drugs such as antihistamines used for asthma work by binding GCPRs

despite the variety all GCPRs share a common structure of 7 transmembrane alpa-helices (aka heptahelical receptors)

adrenaline and glutamate and odorants use GCPRs

many GCPRs are orphans meaning we know their DNA sequence but have not yet identified what ligand they bind to 

how do GCPRs work

GCPRs work through heterotrimeric G proteins (consist of 3 different subunits: alpha (a), beta (b) and gamma (y))

at rest the G protein is bound to GDP

when a signal binds the receptor, the receptor facilitates the switch from GDP for GTP

causes the alpha subunit to break away from the beta-gamma subunits initiating the cascade

how is the switch from GDP to GTP facilitated

the switch from GDP to GTP is facilitated by GEFs (guanine nucleotide exchange factors)

an activated GPCR (with a signal bound) acts as a GEF for its associated G proteins 

historically it was thought that the alpha subunit was the boss of signalling

no we know that the signal bifurcates both the alpha subunit and the beta-gamma complex can independently initiate signalling cascades

what was the first second messenger discovered

cAMP was the first second messenger discovered by Sutherland in the 1970s

produced from ATP by an enzyme called adenylyl cyclase (AC)

how is adenylyl cyclase regulated

AC is regulated in a reciprocal fashion 

a_S (S for stimulatory)  increases cAMP levels 

a_i (i for inhibitory) keeps cAMP levels low

how do most of the cAMPs effects occur

most of the cAMP’s effects occur through protein kinase A (PKA)

when cAMP binds the regulatory parts of the PKA the catalytic subunits are released to go away and phosphorylate other proteins switching them “on”

is signalling transient or constant

signalling must be transient not constant

to turn off the signal, the second messenger must be removed

this is done by enzymes called phosphodiesterases (PDEs) which degrade cyclic AMP into inactive AMP

what pathway does the hormone adrenaline us

adrenaline uses the cAMP pathway

in the heart adrenaline binds beta-adrenergic receptors activating PKA

PKA then phosphorylates proteins that regulate calcium increasing calcium levels and making the heart pump with more force during stress

what is a second major pathway for adrenaline

a second major pathway involves 2 messengers, IP3 and DAG which are both made from the same substrate (a membrane lipid called PI 4,5-bisphosphate)

the enzyme phospholipase C beta (PLC-b) clips this lipid into the 2 messengers

just as a_S activates AC a specific subunit called a_q is responsible for activating PLC-b

describe IP3

IP3 is soluble and diffuses into the cytoplasm to find its receptor on the ER

it  opens a calcium channel allowing the cell to tap into the calcium gradient stored in the ER to do work

what does calmodulin do

calmodulin senses the increase in calcium, it binds 4 calcium ions triggering a massive conformational change that allows it to bind and activate target proteins

what is a key target for calmodulin

a key target for Calmodulin is CamKII

once calmodulin turns the kinase on, CamKII phosphorylates itself, a process called auto-phosphorylation, to fully activate the signalling enzyme 

how are calcium signals terminated

calcium signals are terminated by shuttling the ions back into stores or out of the cell 

the same 3 proteins used to form gradients (PMCA, SERCA and NCX) now act to terminate the signal

difference between IP3 and DAG

unlike IP3, DAG is a lipid and remains in the plasma membrane

its target is protein kinase C (PKC)
PKC is normally floating in the cytoplasm but the calcium released by IP3 binds to PKC and recruits it to the membrane 

once at the membrane PKS “sees” DAG and becomes activated

tyrosine kinase domains

insulin, EGF, PDGF and FGF receptors each have an extracellular domain and an intracellular protein tyrosine kinase domain 

these are “smart” receptors as they are receptors and enzymes in one

they often go wrong in cancer

they respond to growth hormones like FGF and insulin and usually have a single or double transmembrane region

when a stimulus like PDGF binds, 2 receptors come together to form a dimer 

they they phosphorylate their own tyrosine residues (auto-phosphorylation) 

these phosphorylated tyrosines then act as magnets to attract other proteins to the cell surface to begin the work

mitrogen-activated protein kinase (MAPK)

phosphorylated tyrosines recruit Grb-2 (adaptor) that grabs a protein called Sos that then activates Ras (monomeric G-protein)

what is Ras

Ras is a different type of G-protein 

it is a monomeric protein not a heterotrimer 

still used the same GTP switch mechanism 

in the MAPK mechanism Sos acts as the GEF that turns the Ras switch on

MAPK signalling cascade

MAPKKK (Raf) → MAPKK (MEK) → MAPK (ERK)

this is a phosphorylation cascade

one kinase phosphorylates another in sequence

what is FGF

FGF is a key stimulus that works through this MAPK cascade

its physiological role it critical in early development specifically for mesoderm induction in the embryo

guanylyl cyclases

these receptors show homology to receptor tyrosine kinases (RTKs) in that 2 subunits come together

they are “smart” receptors as they combine an extracellular ligand-binding domain with an intracellular cyclase domain that broadcasts the signal in a single polypeptide chain 

(contrasted with GPCRs which have a more segregated mechanism of action)

what is natriuretic

relating to the excretion of sodium 

how are guanylyl cylcase receptors activated

receptors guanylyl cyclases are activated by natriuretic peptides (ligands ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide))

ANP and BNP bond to the extracellular side of the receptor to activate it

what do receptor guanyolyl cyclase produce

receptor guanylyl cyclases produce the messenger cyclic GMP

these receptors take GTP and cyclise it into cGMP (similar to adenylyl cyclase and ATP and cAMP)

this receptor is not attached to the membrane and cannot contact the outside surface directly 

it is activated by nitric oxide (NO)

because NO is a gas it can explain how an intracellular receptor is activated without a membrane-bound component

NO in cell signalling

NO is highly reactive and has a very low half-life

meaning that signalling through NO is localised, occurring only in the vicinity of where it is produced

this contrasts with hormones that circular through the whole body

GMP synthesis

• membrane associated via ANP/BNP (extracellular cues hitting surface receptors which normally would enter the cell)

• soluble via NO (intracellular receptors)

what are phosphodiesterases (PEDs)

phosphodiesterases (PEDs) are the key enzyme responsible for degrading cyclic GMP down to inactivate GMP to dampen the signal

how does cGMP work

just as cAMP works through PKA, cGMP works through protein kinase G (PKG)

in PKG the regulatory and catalytic domains are fused into a single polypeptide chain, whereas they are separate subunits in PKA

what doe NO-cGMP do

nerve terminal (ACh) → endothelial cell (NO synthase) → NO diffusion → smooth muscle cell (soluble guanylyl cyclase) → cGMP → relaxation 

(ACh causes the production of NO gas which diffuses rapidly to the smooth muscle layer of the blood vessel to trigger cGMP production and muscle relaxation)

PKG phosphorylates targets that effectively decrease calcium concentration which induces relaxation (increased calcium causes contraction) 

what does inhibiting PDE do

inhibiting PDE increases cGMP → increases PKG activity → decreases calcium → causes muscle relaxation (pathway earned a nobel prize)

Viagra works this way by inhibiting PDE5 in the cavernous nerve to cause relaxation 

serine/threonine kinases

receptor serine/threonine kinases are the 3rd type of enzyme-linked receptor

they are smart receptors that phosphorylate Serine and Threonine residues distinguishing them from RTKs

best studies examples are for the TGF-beta superfamily, they come in 2 flavours type I and type II

they are activated by ligands of the TGF-beta superfamily (all are secreted proteins): TGF-beta, activin, BMP

unlike small neurotransmitters or gases, these stimuli are full-blown proteins secreted from cells

how do activated TGT-beta receptors signal

activated TGF-beta receptors signal through smad proteins

instead of pure auto-phosphorylation, the type II receptor phosphorylates the type I receptors

once activated the type I receptor phosphorylates smad proteins which are next in the pathway

different TGF-beta ligands phosphorylate distinct smad proteins

TGF-beta/activin/nodal → smad 2/3

BMPs/GDFs → smad 1/5/8

once phosphorylated these smad proteins associate with smad 4 to form a complex that moves into the nucleus

this pathway directly connects the cell surface to transcription 

is much slower than other pathways and vital for early development

TGF-beta signalling (specifically Nodal) works alongside FGF to induce the mesoderm layer in the primordial embryo between the ectoderm (animal) and endoderm (vegetal)

Wnt signalling

Wnt signalling etymology (name comes from merging wingless in flies (wings don't form if inactive) and Int-1 in mice) (focus on canonical pathway)

Wnt (stimulus), Frizzled/Lrp5/6(receptor), Dishevelled (effector), Beta-catenin (transcription factor, most important)

the pathway is interesting as stuff is going on at rest that stops during signalling 

at rest beta-catenin is actively degraded by a 4 protein destruction complex which involves phosphorylation by 3 kinases GSK3 and CK1

genes are blocked by the repressor Groucho

Wnt activated state

when Wnt binds the effector Dishevelled yanks axin out of the destruction complex

without axin the complex fails 

beta-catenin levels rise

it reshuffles into the nucleus and displaces Groucho on genes

Wnt binds receptor → dishevelled yanks axon out of complex → beta-catenin increases → displaces Groucho → transcription 

what does Wnt do

Wnt gradients tell the embryo which part is the head and which is the tail 

in adults Wnt is involved in the constant remodelling and pruning of synapses in the brain

Hedgehod signalling components

Hedgehog (stimulus), Patched (receptor), Smoothened (effector), Ci/Gli (transcription factor) 

stimuli include Sonic, Indian and Desert Hedgehog 

similar to Wnt - involving a secreted protein and a proteolytic degradation mechanism for the transcription factor

what does Hedgehod signalling do

Hedgehog signalling regulates the polarity of segments in the embryo

when mutated the segments are messed up, making the larva look prickly like a hedgehog

what is cell biology (cytology)

the branch of biology studying the structure function and behaviour of cells

what does cell biology seek

cell biology seeks to understand how cells become different during development how they divide and how they move

the movement of a cells leading edge is a highly controlled process involving the organisation and movement of cytoskeletal elements

who coined the term cell

Robert Hooke coined the term “cell” in 1665 after observing thin slides of tree bark  

what he actually saw were the cell walls left behind by dead cells which appeared as irregular subunits or compartments

cells in human body

human body - around 200 cell types, 37 trillion cells total, 96 million die every minute

what happens as the complexity of an organism increases

cells become larger

what is programmed cell death

the process by which the body gets rid of cells to maintain a constant number (Conradt’s research specialty)

smallest and largest cells

the smallest cell is the bacterium Mycoplasms (0.1um) seen here sitting on a much larger mammalian cell

the largest single cell is Valonia ventricosa (“sailor’s eyeballs”) which is one plasma membrane despite being multi-nucleated

bird egg yolks are also single cells

Swan flask experiment

in 1859, Louis Pasteur set out to disprove the spontaneous generation hypothesis (cells arise from non-living material)

in the straight-necked flask, cells reappeared after boiling because spored fell in from the air

the swan-necked flask trapped these spores in a plug of condensation in the neck, keeping the broth sterile

this proved the all-cells-from-cells-hypothesis

where is the genome in prokaryotes

in prokaryotes the genome (DNA) lies freely in the cytoplasm as “electron-dense material” rather than being contained in a nucleus

they generally lack internal compartmentalisation 

some bacteria do have specialisations

when nitrogen is limited Anabaena develops heterocysts to fix nitrogen

these have thick walls to exclude oxygen which would otherwise destroy the nitrogenase enzyme 

vegetative cells remain green for photosynthesis

V:SA

as a cell grows its volume increases faster than its surface area

because food and oxygen must diffuse across the surface, this scaling law limits the size of bacteria

compartments also allow for specialised mixtures, such as the oxygen-free heterocyst

nucleolus

a sub-compartment of the nucleus for ribosomal RNA expression and ribosome assembly

ER

rough ER synthesises proteins, smooth ER synthesises lipids and acts as a calcium store

mitochondria

use oxygen to oxidise to food (sugar) for ATP also involved in signalling and cell death