Module 3: Messengers and Receptors & Mitosis

Long-Range Signalling: Hormones

• hormones are chemical messengers used for long-distance communication between different parts of the body

• this type of signalling is called endocrine signalling

How does endocrine signalling work?

• hormones are produced by endocrine tissues

  • travel from sending to receiving cells via the circulatory system

• secreted directly into the bloodstream

• as they circulate, they bind to specific receptors on target tissues

• their life span ranges from a few seconds to many hours

4 chemical classifications of endocrine hormones

1. Amino acid derivative

2. Peptides

3. Proteins

4. Steroids

2 examples of hormones

• both are types of adrenergic hormones

1. Epinephrine (adrenaline)

2. Norepinephrine

Adrenergic Hormones

• type of endocrine hormone

• produced by the adrenal glands

• stimulates breakdown of glycogen → supply glucose to muscles

• activate flight-or-fight response

  • puts body functions on hold and redirects sources in stressful situations

2 types of adrenergic hormone receptors

1. α-adrenergic receptors

   • binds both epinephrine and norepinephrine

   • located on smooth muscles of visceral organs

   • activates Gq proteins

   • stimulates effector cells

   • cause constriction of blood vessels

2. β-adrenergic receptors

   • binds epinephrine better than norepinephrine

   • located on smooth muscles in hearts, lungs, skeletal muscles

   • activates Gs proteins

     • stimulates cAMP signal transduction pathway

   • relaxes effector cells

   • cause dilation of blood vessels

How do adrenergic hormones function?

• epinephrine binds to beta-adrenergic receptors on liver or muscle cells

  • this activates Gs protein, stimulating adenylyl cyclase

• adenylyl cyclase converts ATP → cAMP (second messenger)

• cAMP activates Protein Kinase A (PKA)

• PKA phosphorylates and activates phosphorylase kinase

  • PKA can also phosphorylate glycogen synthase and inactivate it

• phosphorylase kinase activates glycogen phosphorylase a

  • less active form → more active form

  • leads to an increased rate of glycogen breakdown

• glycogen phosphorylase a breaks down glycogen → glucose-1-phosphate

why do α-adrenergic receptors use IP₃ pathway?

• α-adrenergic receptors stimulate the formation of IP3 and DAG

  • this increases calcium concentration

  • leads to smooth muscle contraction,

    • constricts blood vessels and reduce blood flow

2 other hormone examples

• both are secreted by islets of Langerhans in the pancreas

  1. Glucagon

     • increases blood sugar through glycogen breakdown

  2. Insulin

     • lowers blood sugar by promoting glycogen synthesis into muscle and adipose cells

2 examples of diabetes

• Type I Diabetes

  • body cannot make insulin

    • loss of insulin-producing cells in the islets of Langerhans

    • can be successfully treated with insulin

• Type II Diabetes

  • the body resists insulin

    • it produces it but cells do not respond well

    • cannot be treated with insulin

How does insulin function?

• insulin binds to receptor tyrosine kinases on the cell surface

• this causes auto-phosphorylation of the receptor

• the receptor then phosphorylates IRS-1 (insulin receptor substrate 1)

2 pathways for insulin signalling

• Phosphorylated IRS-1 stimulates 2 different pathways

1. Ras-MAPK Pathway

   • IRS-1 binds GRB2 → Sos, which leads to activation of:

     • Ras

     • → Raf

     • → MEK

     • → MAPK

2. PI3K-Akt Pathway

   • IRS-1 activates PI 3-kinase (PI3K)

   • PI3K converts PIP2 → PIP3

   • PIP3 activates Akt (protein kinase B)

   • Akt increases GLUT4 (glucose transporter) movement to the cell membrane → glucose uptake

   • stimulates glycogen synthesis

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GRB2 → Sos → Ras → Raf → MEK → MAPK

What happens when AKt is activated?

1. Increased Glucose Uptake

   • Akt causes GLUT4 transporters to move to the cell membrane

   • this allows glucose to enter the cell from the bloodstream

2. Increased Glycogen Production

   • Akt activates enzymes that promote glycogen synthase

   • this leads to more glucose being stored as glycogen

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IRS-1 → PI 3-kinase → Akt → GLUT4 → Glycogen synthase

• Pi 3-kinase converts PIP2 → PIP3

• PIP3 activates Akt

Steroid Hormones

• are hydrophobic, so they easily pass through the cell membrane

• inside the cytoplasm, they bind to receptor proteins (mediate the action of steroid hormones)

  • the hormone-receptor complex travels into the nucleus

  • this activates transcription of target genes

• examples: progesterone, estrogen, testosterone

How does gases act as cell signals?

• dissolved gases can sometimes serve as cell signals

• in animals, oxygen and carbon dioxide act as long-range signals in respiration

• nitric oxide acts as a local signal, important for the nervous system

• in plants, ethylene gas signals fruit ripening

Cell Cycle

• when two new cells are formed via cell division of a parent cell

• the cycle ends when the cell divides again

Cell Division

• one parent cell splits into two daughter cells

Before this happens:

• the DNA in the nucleus must be accurately copied

  • this is DNA replication

• the DNA must be evenly shared between the daughter cells

Chromosomes

• formed at the beginning of mitosis

• when chromatin folds and condenses to produce visible chromosomes

• by this point, DNA has been replicated

  • so each chromosome has 2 sister chromatids (identical copies)

  • these sister chromatids are joined together at the centrosome

What are the phases of the cell cycle?

• mitosis is a short part of the cell cycle

• most of the time, the cell is in interphase, which includes:

  • G1 phase

  • S phase

  • G2 phase

• the full cycle takes about 18-24 hours in mammalian cells

Generation Time

• overall length of the cell cycle

Mitotic Index

• percentage of time in M phase (mitosis)

• this takes about 30-45 minutes in mammalian cells

G Phases

1. G1 Phase

   • highly variable depending on cell type

   • this is when the cell grows and decides whether to divide again

   • G₀ → if the cell stops dividing, it enters a resting state

2. G2 Phase

   • shorter and less variable

Terminal Differentiation

• when cells permanently exit the cell cycle (stop dividing)

M Phase

• the cell splits into two daughter cells

M phase includes:

1. Nuclear division (mitosis)

2. Cytoplasmic division (cytokinesis)

S Phase

• the cell replicates its DNA

  • this creates two identical copies of each chromosome

What are the phases of mitosis?

1. Prophase

2. Pro-metaphase

3. Metaphase

4. Anaphase

5. Telophase

Prophase

1. Chromosomes condense

   • DNA coils into visible chromosomes

   • each one has 2 sister chromatids

   • this occurs towards the end of G2

   • cells are in prophase when chromosome is visible

2. Centrosomes move apart

   • these organize microtubules

   • they begin forming the mitotic spindle

3. Asters form

   • star-shaped arrays of microtubules near each centrosomes

4. Centrioles (in animal cells)

   • located inside centrosomes

   • help organize spindle formation

Centrosomes

• found near the nucleus

• function as microtubule-organizing centers (MTOC)

Pro-Metaphase

1. Nuclear envelope breaks down

   • this lets spindle microtubules reach the condensed chromosomes

2. Centrosomes reach opposite sides of the cell

   • this completes spindle formation

3. Spindle microtubules connect to chromosomes

   • they attach at a special region called the kinetochore, located at the centromere

Kinetochore

• the region where spindle microtubules attach to chromosomes

• this is located at the centromere

Structure:

• Inner kinetochore

  • CCAN forms this inner part

  • bind directly to the centromeric DNA

  • contains CENP and other proteins

• Outer kinetochore

  • KMN network forms this outer part

  • attaches to the plus end of microtubules

Metaphase

• chromosomes are fully condensed

  • they are lined up at the at the middle of the cell → metaphase plate

• sister chromatids of each chromosome are being pulled in opposite directions via spindle fibers

• congression → series of movement that pull chromosomes to the center of mitotic spindle

• drugs like colchicine can stop cells during metaphase, by disrupting spindle function

Anaphase

• shortest phase of mitosis

• sister chromatids suddenly separate

  • they move toward opposite poles

Telophase

• daughter chromosomes arrive at the spindle poles

• they uncoil into loose chromatin (interphase-like)

• the nuclear envelope reforms

• nucleoli reappears

• during this period, cytokinesis begins

Are all hormones perceived in the plasma membrane?

no, not all hormones are perceived in the plasma membrane

Hormones with plasma membrane receptors:

• these hormones are hydrophilic (water-soluble)

• cannot cross the plasma membrane

• examples: epinephrine, norepinephrine, insulin, glucagon

Hormones with intracellular receptors:

• these hormones are hydrophobic (lipid-soluble)

• they can diffuse through the membrane

• examples: steroid hormones

Mitotic Spindle

• microtubule-based structure that ensures equal separation of daughter chromatids during mitosis

• microtubules have inherent polarity:

  • minus end → anchored at the centrosome

  • plus end → grows outward and connects to chromosomes

3 types of microtubules

1. Kinetochore MTs

   • pull chromosomes toward the center of the cell

2. Polar MTs

   • overlap with MTs from the opposite pole

3. Astral MTs

   • shorter and forms asters at each pole

   • some interact with proteins lining the plasma membrane

Spindle Assembly & Chromosome Attachment

• microtubules grow from centrosomes, which serve as MTOC

• the minus end of a microtubule stays anchored at the centrosome

• the plus end grows outward, towards chromosomes

• during late prophase, microtubule growth occurs rapidly

  • this includes initiation of new MTs as centromere increases

• the nuclear envelope breaks down, allowing MTs to reach chromosomes

  • the MT becomes a kinetochore MT

• each kinetochore usually connects to MTs from opposite spindle poles

Polar MTs Chromosome Attachment

• polar microtubules from opposite poles grow toward each other

• they overlap in the middle and are stabilized by cross-linking proteins

How do chromosomes move during mitosis?

• chromosomes move through congression

  • pulls them to middle of mitotic spindle

  1. Cytoplasmic dynein pulls chromosomes toward spindle poles

     • this occurs on kinetochore MTs

  2. Kinesin CENP-E pushes chromosomes away from spindle poles

     • this occurs on kinetochore MTs

  3. Chromokinesins (kinesin-4 & kinesin-10) push chromosomes

     • this occurs on polar MTs

Microtubule activity during anaphase

Anaphase is split into two overlapping processes:

• depending on the cell type, Anaphase A and B can happen simultaneously or one after the other

• Anaphase A → Chromosomes move

  • as kinetochore MTs shorten, the chromosomes are pulled toward spindle poles

• Anaphase B → Poles move apart

  • polar MTs lengthen, pushing the spindle poles farther apart

Cytokinesis

• after chromosomes are separates, cytokinesis splits the cytoplasm into two daughter cells

• begins late in anaphase or early in telophase

• some cells skip cytokinesis after nuclear division

  • this results in a multi-nucleated cell → syncytium

  • this can be permanent or temporary

Cytokinesis in Animal Cells

• the cell surface begins to pucker, forming a cleavage furrow

• a ring made of actin microfilaments (contractile ring) forms beneath the membrane during early anaphase

• as cleavage progresses, the ring tightens around cytoplasm

• the narrow connection becomes a thin stalk

  • eventually snaps apart, fully separating the two daughter cells

Cytokinesis in Plant Cells

• does not have tightening of contractile ring due to cell wall

• instead, the plant builds a new cell wall and plasma membrane between the two daughter nuclei

  How it works:

  • small vesicles from Golgi align at the center of the cell

    • this occurs during late anaphase to early telophase

  • these vesicles carry polysaccharide and glycoproteins to help build the new wall

  • guided to the spindle equator via phragmoplast → an array of MTs derived from polar MTs

  • fuse together, forming a cell plate

    • the cell plate extends outwards from the cell wall, dividing the cell into two

Bacterial Binary Fission

• bacteria divides in a different manner from eukaryotic cells

  • they divide via binary fission

• they use a protein called FtsZ → similar to tubulin

• FtsZ forms a ring inside the bacteria, where the cell will split

  • this helps pinch it into two daughter cells

• some eukaryotic organelles also use FtsZ to divide

3 variations of the cell cycle

• different cells go through the cycle at different speeds

• there are 3 main ways the cycle varies:

  1. overall length of the cycle

  2. relative length of time spent in each phase

  3. how tightly linked mitosis and cytokinesis are

• cells adjust their cycle to meet their needs

How does cell cycle length vary by cell type?

• some cells divide constantly to replace cells that are regularly lost or damaged

  • examples: sperm-forming cells, stem cells

• others divide very slowly, or not at all

  • examples: mature nerve or muscle cells

• some cells only divide when stimulated

  • examples: in response to injury or specific signals

2 variations in generation time

• variations in generation time are based on the length of G1

• S and G2 phases can also vary, but less so

Two main patterns:

1. Slow-dividing cells

   • spend a long time in G0

     • resting phase branching off from G1

     • can last days, months or years

2. Fast-dividing cells

   • have a short G1 or may even skip G1 entirely

Why does the cell cycle need to be regulated?

the cell cycle is controlled at key transition points to:

1. Make sure each step happens in the correct order

2. Ensure that each phase is completed before the next one begins

3. Respond to external conditions

Restriction Point (G1-S Transition)

• first major checkpoint in the cell cycle, near the end of G1 phase

  • G1 → S progression

  1. called Start in yeast

  2. called the restriction point in animal cells

• the cell determines if conditions are right to continue

• if the cell passes this point, it is committed to entering S phase

  • this is influenced by presence of extracellular growth factors

G2-M Transition

• At the G2 → M boundary

  • the commitment is made to enter mitosis

• in some cell types, the cell can be arrested in G2, similar to how it can rest in G₀

  • example: frog eggs

• in most cells, the G1 arrest is more common point of control

Metaphase-Anaphase Transition

• this checkpoint happens between metaphase and anaphase

• commitment is made to move the two sets of chromosomes into the new cells

• before the cell proceeds to anaphase, all the chromosomes must be properly attached to the spindle

What are the mechanisms regulating the cell cycle?

• CDKs (cyclin-dependent kinases) regulate the cell cycle

  • enzymes that control the cell cycle by phosphorylating target proteins

  • only active when bound to regulatory protein → cyclin

• Protein phosphatase also help turn off cell cycle signals

  • enzyme that remove phosphate groups

How does cell cycle phases affect cyclin concentration?

• cyclins are produced and degraded in a specific order to regulate different phases:

  1. Mitotic cyclins

     • required for the G2 → M phase transition

     • bind mitotic CDKs to form MPF (mitosis-promoting factor)

  2. G1 cyclins

     • help the cell pass the G1 restriction point or Start

     • binds G1 CDKs to push the cell toward S phase

  3. S cyclins

     • trigger DNA replication in S phase

How are mitotic CDKs regulated?

1. Cyclin availability

2. CDK phosphorylation

Cyclin Availability

• mitotic CDK has constant concentration throughout cell cycle

• CDK is only active when bound to mitotic cyclin

  • mitotic cyclin is not always present in adequate amounts

• cyclin levels increase during G1, S, and G2

• when cyclin reaches a threshold at the end of G2

  • it activates the mitotic CDK to trigger mitosis

• halfway through mitosis, cyclin is destroyed

  • this turns CDK off

CDK Phosphorylation

• even when CDK binds to cyclin, it’s still inactive at first:

• Step 1: Add “off” phosphates

  • inhibitory kinases add 2 phosphates to the CDK → keeps it inactive

• Step 2: Add “on” phosphate

  • activating kinase adds a third phosphate, but it’s still inactive

• Step 3: Remove the brakes

  • phosphatase enzyme removes the 2 inhibitory phosphates

    • now CDK becomes active

• once active, the CDK can stimulate more phosphatase

What is the function of CDKs?

once activated, the CDK-cyclin phosphorylates:

1. Lamin

   • causes lamina breakdown

   • destabilizes nuclear envelope

2. Condensin

   • involved in chromosome condensation

3. Microtubule-associated proteins

   • facilitates mitotic spindle assembly

4. Anaphase-promoting complex

Anaphase-Promoting Complex

• mitotic CDK-cyclin helps activate APC by phosphorylation

• APC acts as a ubiquitin ligase

  • it tags specific proteins w/ ubiquitin

  • targets them for destruction

• before anaphase, securin blocks separase protein

  • inhibitor that splits sister chromatids

  • when APC destroys securin, separase becomes active

• separase cuts cohesins

  • adhesive protein that holds sister chromatids together

• sister chromatids separate, starting anaphase

• APC also destroys mitotic cyclin

  • inactivates CDKs, helping the cell exit mitosis

5 cell cycle checkpoints

1. G1-S

   • Rb and E2F

2. S phase

   • replication licensing

3. G2-M

   • DNA replication checkpoint

4. M phase

   • mitotic spindle checkpoint

5. DNA damage

   • occur throughout the cell cycle

Checkpoints

• cells use checkpoints to make sure each phase is completed properly before the next one begins

  • if cells proceed to next without completing each step, daughter cells can become abnormal

G1-S Checkpoint

• the cell needs to initiate gene transcription before it can enter S phase

  • E2F protein turns on these genes, but blocked by Rb

• when Rb is bound to E2F, the E2F molecule is inactive

  • the cell cannot enter S phase

• growth factors activate G1 CDK-cyclin

  • this complex phosphorylates Rb

  • causes Rb to release E2F

  • free E2F turns on genes needed for DNA replication → the cell can now enter S phase

S Phase Checkpoint

• this checkpoint ensures DNA is copied only once before the cell divides

• each origin of replication is “licensed” to start copying DNA

• once it’s used, the license is removed → cannot be reused in the same cycle

• prevents over-replication or missing sections of DNA

G2-M Checkpoint

• makes sure DNA synthesis is completed before the cell exits G2 and begins mitosis

M Phase Checkpoint

• this checkpoint makes sure all chromosomes are properly attached to the spindle, before entering anaphase

DNA Damage Checkpoints

• these checkpoints check for DNA damage at multiple stages:

  • Late G1 → before DNA is copied

  • S phase → while DNA is copied

  • Late G2 → before mitosis begins

• if damage is found, the cell blocks CDK-cyclin activity (which drive the cell cycle forward)

What happens when cell cycle regulation fails?

if checkpoints do not work properly, cells may:

• divide uncontrollably → cancer

• pass on damaged or mutated DNA

• become abnormal and harmful

damaged or diseased cells need to be eliminated

• this can occur via apoptosis or necrosis

2 types of cell death

1. Apoptosis

   • a programmed cell death without damaging surrounding cells

   • occurs via activation of caspases enzyme

     • produced as inactive precurser → procaspases

     • the precursor is cleaved and turned into active caspases

2. Necrosis

   • uncontrolled cell death, caused by injury → infection, toxins, trauma

   • the cell swells, bursts and releases its contents into the surrounding area