Biology of Cells Quiz 2

Functions of membrane

1. Define boundary

2. Sites of specific function

3. Regulation of solute transport

4. Signal detection & transmission

5. Cell-cell communication

Carbohydrates in cell membrane

• recognition patterns for cells & organisms

• Glycoprotein/glycolipid depending on what it is attached to

• carbohydrate chains form glycocalyx (used in cell recognition)

Cholesterol in cell membrane

Affects membrane rigidity

Lipid composition of cell membrane

• Asymmetric composition of outer vs inner membrane (e.g. glycolipids only on outer)

• Movement via lateral diffusion

• Little/no transverse diffusion from outer/inner membrane to the other

  • Flippases flip lipids from outer to inner and vice versa

Selective permeability depends on…

Size, charge, polarity

Small & less polar molecules can more easily diffuse

Can freely diffuse across membrane

• O2 & CO2

• Nitrogen

• Steroid hormones

• Ethanol

• Very small nonpolar molecules

Can diffuse slowly across membrane

• Water

• Urea

• glycerol

• small, uncharged molecules

Needs transport protein to diffuse across membrane

• Ions

• glucose & other sugars

• amino acids

• proteins & macromolecules

2 types of membrane proteins

• Integral

  • embedded permanently in membrane

  • can be transmembrane: exposed to environment on both sides of membrane

• Peripheral

  • temporarily attached to surface of membrane

Functions of membrane proteins

• structural support

• transport

• enzymatic control of cell processes

• receptors for hormones & other molecules

• “self’ markers for immune system

4 major types of tissue in animals

• epithelium

• connective tissue

• nervous tissue

• muscle tissue

Cell adhesion

Different cell-adhesion molecules (CAMs) allow cells to attach to each other or to ECM

Cell-adhesion molecules (CAMs)

• many are transmembrane proteins

• continuously recycled

• classes:

  • immunoglobulin super family (IgSF)

  • cadherins

  • selectins

  • integrins

Homophilic and heterophilic interactions of CAMs

Homophilic:

• Cells with identical receptors interact

• In many IgSF and cadherin membranes

Heterophilic:

• Cells with different receptors interact

• In selectins & integrins

Lectins

• Carbohydrate-binding proteins produced by many animals & plant cells

• Promote cell-cell adhesion by binding to specific sugars on glycoproteins or glycolipids on outer cell surface

• Selectins are type of lectin that mediates cell-cell adhesion

Integrins

• Attach to ECM proteins to mediate cell adhesion & migration

• Integrins connected to actin filaments via linker proteins to facilitate motility by forming focal contacts/adhesion

• dynamic assembly & disassembly of focal adhesions allow WBCs to migrate along connective endothelium following cellular signals to damaged biological tissues

Cell-cell junctions

• Helps join cells in long-term associations to form tissues & organs

• specialized structures where 2 cells come together are called cell-cell junctions

• links cells together to form tissue & enable cells to function as unit

Types of cell-cell junctions & functions

• tight:

  • prevents leakage of extracellular fluid between cells

  • leaves no space between membranes of adjacent cells

  • forms continuous belt around apical ends of lateral surfaces of each cell in epithelial cells

• anchoring/adhesive: fasten cells together into sheets

• gap:

  • channels that allow electrical and chemical communication between cells

  • located in regions where plasma membranes of cells are aligned and brought into contact

  • formed by assembly of connecting channels made of connexins (channels not always open)

Endomembrane system

• group of membranes & organelles in eukaryotic cells that work together to modify, package, transport lipids and proteins

• includes:

  • nuclear envelope

  • ER

  • golgi apparatus

  • lysosomes

  • plasma membrane

Endoplasmic reticulum

• present in almost every eukaryotic cell

• tubular membranes (smooth ER) + cisternae (rough ER)

• internal space of ER = lumen

• ER is continuous with other membranes in cell

• rough & smooth ER luminal space continuous

Proportion of rough and smooth ER in eukaryotic cells

• rough ER > smooth ER in cells producing proteins

• smooth ER > rough ER in cells producing lipids and steroid hormones

Rough ER

• studded with ribosomes on cytoplasmic side of membrane

• synthesize proteins destined for export from cell

• functions:

  • manufacture lysosomal enzymes

  • manufacture secreted proteins

  • manufacture integral membrane proteins that stay embedded in membrane

  • initial glycosylation as assembly continues (continued in golgi)

  • recognition and removal of misfolded polypeptides (exported from ER for degradation by cytosolic proteasomes)

  • assembly of multimeric proteins (2 or more polypeptide chains held together covalently)

Cotranslational import

• all protein synthesis begins on free ribosomes

• if polypeptide is destined for export from cell, free ribosome attaches to ER membrane early on in translation

• newly forming polypeptide chain is transferred across ER membrane while synthesis occurs → polypeptide chain is inside ER lumen

• Steps:

  • ER signal peptide (AA sequence) recognized by signal recognition particle (SRP) in cytosol

  • SRP binds to sequence and inhibits translation

  • Ribosome + mRNA + partially translated protein brought to ER and SRP binds to SRP receptor at surface of ER

  • Ribosome attached to translocon channel

  • SRP dissociates (returns to cytosol) and ribosome locks onto translocon channel

  • As translation continues, polypeptide chain is inserted into ER lumen through channel

  • Signal peptidase (enzyme) cuts initial signal peptide from growing polypeptide chain

  • Translation finishes & releases polypeptide chain into ER lumen

Posttranslational import

• ribosomes remain free if producing polypeptides destined for cytosol, mitochondria, peroxisomes, nucleus

• after translation complete, polypeptides released from ribosomes & remain in cytosol or taken up by appropriate organelle by posttranslational import

• specific targeting signals on proteins required for posttranslational import

• nucleus: happens via nuclear pores (different from other organelles)

Smooth ER

• synthesis of lipids & steroids

• central role in biosynthesis of membranes

• function varies depending on cell type:

  • liver: contains glucose-6-phosphatase to break down stored glycogen & has enzymes for detoxification

  • endocrine glands: synthesis of steroid hormones

  • muscle: sarcoplasmic reticulum (specialized smooth ER) stores & releases calcium ions to trigger muscle contraction

Golgi apparatus

• closely related to ER in proximity & function

• made up of cisternae

• cis golgi network is entry point, trans golgi network is exit point for vesicles

• process & package secretory proteins + synthesis of complex polysaccharides

  • accepts vesicles that bud off ER

  • sorts, modifies, processes contents of vesicles from ER

  • completes glycosylation of secretory and membrane proteins (started in ER)

  • processed proteins move to other locations in cell through vesicles that bud off golgi

Glycosylation important for…

• proper folding

• protein function

• appropriate targeting

Protein trafficking

• Proteins synthesized by rough ER either directed to intracellular locations or incorporated into plasma membrane or exported out of cell

• Protein has specific tag targeting protein to transport vesicle that carries it from one cellular location to another

• Tag can be amino acid sequence, oligosaccharide side chain, hydrophobic domain, etc

Signal hypothesis

• Proteins need signal sequence to sort to an organelle

• Sequence specifies protein destination

Lysosomes

• part of endomembrane system (receives vesciles from golgi)

• filled with digestive enzymes

• Steps

  • fuse with endosomes (membrane-bound organelle that sorts materials, contains vesicles) that contain food/bacteria/dead cells

  • primary lysosome: only contains digestive enzymes (stored enzymes, no material to digest)

  • secondary lysosome: contains partially digested contents of food vacuole or worn-out organelles (enzymes + digested material)

  • residual body: filled with waste which can be expelled through exocytosis

• Other functions:

  • autophagy: digests worn-out/damaged organelles and proteins in cell

  • apoptosis: programmed cell death (lysosome spills its own contents to kill cell)

Peroxisomes

• not part of endomembrane system (don’t receive vesicles from golgi)

• contains different enzymes specific oxidative reactions

• peroxins: peroxisomal proteins (assembly & maintenance of peroxisomes)

• found in most cells but most numerous in liver for detoxification

• eliminate free radicals by producing hydrogen peroxide as byproduct

• contains catalase (enzyme) which converts hydrogen peroxide into water and oxygen

Vesicular transport

• secretion can be constitutive (continuous & unregulated) or regulated by signals

• core parts:

  • exocytosis

  • membrane fusion

  • endocytosis

  • phagocytosis & autophagy

  • organelle transport

Exocytosis

• ER → golgi → plasma membrane

Regulating transport: GLUT4 & insulin

• regulates glucose uptake by physically moving GLUT4 transporters instead of changing concentration gradient of glucose

• Concentration gradient never changes, what changes is number of transporters

• insulin binds to receptors = signaling for vesicles carrying GLUT4 to move to surface

• more GLUT4= faster glucose uptake

• decreased insulin = endocytosis moves transporters back inside

Membrane fusion

• anterograde transport: COPII-coated vesicles go from ER to golgi

• retrograde transport: COPI-coated vesicles return from golgi to ER and between golgi compartment

• clathrin-coated vesicles transport proteins between trans-golgi network, endosomes and plasma membrane

Anterograde transport

1. Sar1 membrane binding, GTP exchange

   • Sar1-GDP (GTPase) interacts with Sec12 (GEF) on ER membrane

   • Sec12 facilitates exchange of GDP for GTP

   • Binding GTP → conformation change on Sar1, exposes hydrophobic N-terminus which inserts directly into ER membrane

2. COPII coat assembly

   • Sar1-GTP recruits Sec23/24 (inner coat protein complex)

   • Sec23/24 recruits cargo (sec24 is subunit that directly binds cargo)

   • Sec13/31 (outer coat protein complex) binds → deforms membrane and leads to vesicle budding

3. GTP hydrolysis

   • after budding, Sar1 hydrolyzes GTP to GDP

   • Sec23 is GTPase-activating protein (GAP) that triggers this step

4. Coat disassembly

   • GTP hydrolysis causes Sar1 to change shape & release hydrophobic tail from membrane

   • Without anchor support, COPII coat complex disassembles into cytosol → forms uncoated vesicle ready to fuse

Retrograde transport

1. Activation

   • Arf1-GDP (GTPase) in cytosol interacts with Gea (GEF) on golgi membrane which exchanges GDP for GTP

2. Arf1-GTP exposes lipid anchor which inserts into golgi membrane

3. Arf1-GTP recruits inner layer coat complex (β/γ/δ/ζ-COP) and outer coat protein complex (α/β′/ε-COP) to capture cargo proteins

4. Coat complex polymerizes on membrane (come together) → bend and bud off as COPI-coated vesicle

Clathrin-coated vesicles

• transport specific molecules and proteins within cell

• originate from different compartments

• marked by different cargo adaptor protein complexes found at different donor membranes

Vesicle fusion

• Rab proteins are small GTPases that act as specificity factors

  • mediate tethering: rab protein on vesicle interacts with tethering protein on target membrane

  • direct cargo traffic & initial docking: vesicle moves closer as v-SNARE and t-SNARE begin interacting

• SNARE proteins: family of short helical transmembrane proteins

  • vesicles carry v-SNAREs and target membranes have complementary t-SNAREs (fusion specificity)

  • formation of v+t SNARE complex stimulated by Rab-GTP

  • High energy bond brings lipid bilayers together to facilitate fusion

  • squeeze two lipid bilayers together to physically induce membrane fusion → successful delivery of cargo protein

• Botox works by destroying SNARE protein complex required for exocytosis

Endocytosis

• plasma membrane → early endosome → late endosome → lysosome

• can be nonspecific or specific

• virus hijack endocytosis mechanism to enter cells

• functions:

  • receptor signalling

  • migration

  • nutrient uptake

  • receptor downregulation

  • neurotransmission

  • pathogen entry

• types:

  • phagocytosis: “cell eating” (engulfs large particles)

  • pinocytosis: “cell drinking” (engulfs extracellular fluid)

  • receptor-mediated endocytosis (very specific, usually mediated by clathrin)

Clathrin-mediated endocytosis

• AP2 (adaptor protein complex) binds to cargo at inner surface of membrane

• clathrin triskelion binds to AP2 and polymerizes which forces plasma membrane to bend inward → forms membrane invagination

• dynamin (GTPase) assembles as collar at neck of vesicle and pinches off vesicle into cytoplasm

Phagocytosis

• bulk transport of large extracellular substances into cell

• mostly used by phagocytic immune cells

• phagocytosis: pseudopod extensions mediated by actin polymerization engulfs and fuses together to form food vacuole

• lysosome provides enzymes to break down material so food vacuole fuses with lysosome

3 pathway to degradation in lysosome

• phagocytosis

• endocytosis

• autophagy

autophagy

• regulated, destructive mechanism to disassemble unnecessary or dysfunctional components

Fast axonal movement

• transport of vesicles and organelles along microtubules

  • proteins & neurotransmitters produced in cell body must be transported to nerve ending

• microtubules provide organized set of tracks for transport of variety of organelles & vesicles

• inbound (retrograde transport)

  • movement towards minus ends of microtubule

  • mediated by cytoplasmic dynein

• outbound (anterograde transport)

  • movement towards plus ends of microtubule

  • mediated by kinesin I

• motor proteins use ATP to provide energy for movement

Kinesin

• 3 parts:

  • globular head region (attaches to microtubules, involved in ATP hydrolysis)

  • coiled helical region

  • light-chain region (attaches to cargo proteins)

Transport proteins make following exchanges selective:

• take in oxygen, glucose, amino acids, ions

• export CO2, urea, other waste

• balance water and ions to hold cell volume steady

• respond to hormones and neurotransmitters

Cell transport types

• passive

  • high to low conc

  • no saturation if no proteins involved (rate keeps rising with gradient)

  • simple diffusion, facilitated diffusion, osmosis

• active

  • saturation occurs (rate can plateau)

  • primary pumps, organelle proton pumps

• bulk

  • bypass bilayer by carrying molecules inside vesicles

  • requires ATP

  • endocytosis, exocytosis, membrane recycling

Passive transport

• osmosis

  • hypotonic: water enters faster → cell swells and bursts

  • isotonic: water moves in & out at equal rates

  • hypertonic: water leaves faster → cell shrinks

• facilitated diffusion

  • needs proteins (channels & carriers)

  • no energy needed

  • saturation happens

Transport proteins

• ion channels

  • selectively allow molecules through

  • voltage-gated: opens when membrane potential changes

  • ligand-gated: open when neurotransmitter binds

  • mechanically-gated: open when membrane physically changes

• carrier proteins

  • specific to solutes

  • binding site never open to both sides at once so slower than channels

Active transport

1. ATP hydrolysis changes shape of protein pump

2. Primary transport: pump pushes ions against gradient

3. Stored ion gradient

4. Secondary transport: carrier spends stored gradient

Fick’s law

• describes how molecules move from high concentration to low concentrations

• flux = permeability coefficient of membrane x available SA x concentration diference (J=PAC)

• affecting factors:

  • gradient: steeper = larger flux

  • SA: larger = larger flux

  • thickness: thinner barrier = faster diffusion

  • temperature: warmer = faster diffusion

  • molecule size: smaller = faster diffusion

  • lipid solubility: more soluble = higher permeability coefficient

Signal transduction

• how cells convert info into biological responses

• ability of cell to respond to ligand-receptor binding by altering its behavior or gene expression

• cells sense environmental cues, signals from other individuals, signals from other cells

• conversion of one type of signal (extracellular) into another type of signal (intracellular)

• key is crossing boundaries of cell

• ligand binding may trigger production of additional molecules in receiving cell (second messengers)

Cell communication

• only cells with suitable receptors can respond

• reception → transduction → response

  • reception: ligand binds to receptor which changes conformation

  • transduction: proteins & second messengers relay signal

  • response: enzymes/channels/gene regulation changes, cell behavior altered

Ligand

• primary messenger

• may bind receptor on surface or inside target cell

  • cell surface receptor: ligand does not need to enter cell they affect (polar ligands cannot cross lipid bilayer)

  • intracellular receptor: respond to hydrophobic ligand molecules that can cross plasma membrane

Molecular switches

• control cell signaling

• 2 types:

  • phosphorylation switch

    • kinase: transfers phosphate from ATP to protein

    • phosphatase: removes phosphate from protein

    • phosphorylation can either activate or inactivate protein

  • GTPase switch

    • GDP-bound = off state

    • GTP-bound = on state

    • binding of GTP turns switch on (GDP is replaced by new GTP, it is not phosphorylated)

    • hydrolysis to GDP turns switch off

Animal schemes for signaling

methods differ by distance and speed

• endocrine: signaling molecule released from site distant from target

• paracrine: signaling molecule targets nearby cells (short range)

• autocrine: signaling molecule targets same cell

• juxtacrine (contact-dependent): membrane-attached signal targets adjacent cell

• synaptic signaling: mainly used by nerve cells

Cell response

• depends on presence of receptors & combination of signals

• single receptor can activate multiple pathways or multiple pathways can converge onto same second messenger

• different cells respond to same signal in different ways

  • different receptors = different cell responses

    • Epinephrine to α receptor on intestinal blood vessel = vessel constricts

    • Epinephrine to β receptor on skeletal muscle blood vessel = vessel dilates

  • different intracellular proteins = different cell responses

    • Epinephrine to β receptor on skeletal muscle blood vessel = vessel dilates

    • Epinephrine to β receptor on liver cell = glycogen breaks down and glucose released from cell

• cellular responses can be fast or slow

  • fast usually alter functions of existing proteins

  • slow usually alters gene express

Specificity of receptor & ligand binding

• one/few ligands to one/few receptors

• binding site on receptor fits ligand very closely

• necessary amino acid side chains on receptors positioned to form chemical bonds with ligand

Affinity of receptor & ligand binding

• relationship between concentration of ligand in solution and number of receptors occupied

• dissociation constant (K_d) is concentration of free ligand needed to produce state in which half the receptors are occupied

• receptors with high ligand affinity have low K_d (easier to bind = lower [free ligand] needed for half of receptors to be occupied)

• possible to make synthetic ligands that bind more tightly or selectively than biological ligands

  • agonists: drugs that activate receptor they bind to

  • antagonists: bind receptors without triggering change (prevents natural messengers from binding & activating receptors)

4 general groups of receptors

• ligand-gated ion channels

• g-protein coupled receptors

• receptor tyrosine kinase

• intracellular hormone receptors

G-protein coupled receptor structure

• 7 transmembrane alpha helices connected by alternating cytosolic & extracellular loops

• extracellular regions bind ligands

• intracellular loops bind g-proteins

G-protein coupled receptors (GPCR)

• ligand binding → change in receptor conformation → activates particular G protein

• activated G protein binds to target protein → alters target’s activity

G proteins

• GTPases that act like molecular switches whose on/off state depends on whether bound to GTP or GDP

• 2 classes:

  • large heterotrimeric G proteins (have alpha, beta, gamma subunits)

  • small monomeric G proteins

Mechanism of GPCR

1. Signaling molecule binds to GPCR

2. GPCR binds to heterotrimeric G protein → stimulates release of GDP and binding of GTP to alpha subunuit

   • ligand-bound receptor acts as GEF

3. activated alpha subunit dissociates from beta & gamma subunits and activates second messengers

G proteins functions

• heterotrimeric G proteins activate second messengers

• regulate calcium ion

• betta/gamma subunits can also transduce signals

GPCR generation of cAMP

• occurs in response to epinephrine in muscle for glycogen breakdown

• GTP-bound G_alpha activates adnylate cyclase

• adenylate cyclase generates cAMP from ATP

• cAMP activates protein kinase A (PKA)

• PKA phosphorylates protein substrates

GPCR activation of phospholipase C

• during histamine signaling

• GTP-bound G_alpha activates phospholipase C (PLC)

• PLC cleaves PIP₂ into DAG and IP₃

• IP₃ causes Ca²⁺ to release from ER

• DAG and Ca²⁺ activate protein kinase C (PKC) to phosphorylate proteins

Receptor tyrosine kinases (RTKs)

• growth factors usually signal through RTKs

• RTK signaling often results in increased cell growth, proliferation, survival (not intrinsic to any signal/receptor)

Mechanism of RTKs

• ligand binding to receptor promotes pairing (dimerization) of RTK monomers

• dimerization activates kinases so each receptor kinase phosphorylates tyrosine residues on its partner receptor (other receptor in pair, autophosphorylation)

• after phosphorylation, tyrosine residues become phosphotyrosine sites which act as docking sites to recruit intracellular signaling proteins

Phosphorylation can change protein function

• causes conformational change which affects activity

• creates binding site for another protein

• blocks binding of another protein

Structure of RTKs

• single polypeptide chain with one transmembrane segment

• extracellular part contains ligand-binding domain

• cytosolic side has tyrosine kinase domain

Activation of RTK

• ligand binding = signal transduction initiated

• causes aggregation of RTKs

EGFR activation

• type of RTK

• steps:

  1. receptor dimerization

     • EGF binds = conformation change = receptor dimerizes

  2. dimerization = kinase domain activated = use ATP for autophosphorylation

     • phosphorylation provides positive feedback that further activates kinase domain

  3. recruitment of signaling proteins

     • with EGF bound and receptor dimerized, kinase activity increases

     • RTK ICD phosphorylation → recruitment of proteins with SH2 and PTB domains (induced proximity)

     • these two domains bind to phosphorylated tyrosines on receptor

RTK recruitment of signaling proteins: Induced proximity

• bringing together two proteins that otherwise wouldn’t interact much

• activation driven by change in localization (protein moves from one place to another) not allosteric/covalent modifications

• Key proteins:

  • GRB2: cytosolic, contains SH2 domain (binds phosphotyrosine sites on activated RTK) & SH3 domain (binds SOS)

  • SOS: cytosolic, acts as GEF for Ras

  • Ras: PM-bound, small GTPase

• GRB2 brought to membrane by phosphotyrosine on RTK → SOS brought to membrane by GRB2 → activates Ras

Ras

• small GTPas (not kinase)

• binds to effector proteins including 2 major branching pathways:

  • Raf

    • cytoplasmic protein kinase

    • stimulates cell proliferation

  • PI3-kinase

    • membrane-associated lipid kinase

    • stimulates cell survival & growth via membrane recruitment of AKT kinase

Downstream of Ras: MAPK signaling cascade

• activated by growth factors or cytokines

• Step-by-step phosphorylation chain where each kinase uses ATP to phosphorylate and activate the next

  • activated RTK recruits GRB2 and SOS

  • SOS promotes Ras GDP→GTP exchange

  • MAPKKK

    • activated by activated Ras-GTP (which is why it is downstream of Ras)

    • Raf in EPK pathway

  • MAPKK

    • activated by MAPKKK

    • Raf phosphorylates MEK in ERK pathway

  • MAPK

    • activated by MAPKK

    • MEK phosphorylates ERK in ERK pathway (regulated downstream targets)

• activated MAPK phosphorylate 2 main targets to cause cellular response:

  • cytoplasmic targets (proteins, cause immediate changes to protein activity)

  • transcriptional factors (cause changes in gene expression)

MAPK signaling into nucleus

• phosphorylation of MAPK functions:

  • promote nuclear import

  • activates transcription factors via phosphorylation (growth or cell cycle regulators)

• Signaling cascade:

  • ligand activates RTK through dimerization & transphosphorylation of ICD

  • activated RTK activates Ras through GRB2-SOS recruitment

  • Ras-GTP activates Raf (MAPKKK)

  • Raf phosphorylates MEK (MAPKK)

  • MEK phosphorylates MAPK

  • MAPK enters nucleus & phosphorylates transcription factors

  • increases genes required for growth & proliferation

Insulin signaling

• rapid & long-lasting effects on many cells

• insulin binds to RTKs which have 2 alpha and 2 beta subunits (exception, usually RTKs have singular subunit)

• when insulin binds to receptor, beta subunits phosphorylate insulin receptor substrate I (IRS-1)

• phosphorylated IRS-1 stimulates 2 different pathways:

  • recruit GRB2 and activate the Ras pathway

  • bind phosphatidylinositol-3-kinase (PI3-kinase) which converts PIP₂ into PIP₃

Insulin signaling regulates glucose uptake: PI3K-AKT pathway

• PIP₃ binds protein called AKT (aka protein kinase B)

• activation of AKT has 2 important consequences:

  • movement of GLUT4 to PM to allow glucose uptake

  • phosphorylation of glycogen synthase kinase 3 (GSK3) to increase its activity and increase amount of active form of glycogen synthase to enhance glycogen production

• Antagonist enzyme of PI3-kinase is PTEN (phosphatase that removes phosphate from PIP₃ and prevents activation of AKT)

steroid hormones

• lipid signaling molecules

• receptors primarily act in nucleus not cell surface

• receptors mediate action of steroid hormones

• hormone enters target cell & binds receptor → triggers cascade of events that activate/inhibit transcription target genes

Cell cycle

• functions:

  • reproduction: produce new unicellular eukaryotic organism

  • development: increase cell number in growing tissues

  • renewal & repair: replace lost cells & repair damage

• cell growth increase size; cell proliferation increases cell number

• 2 major phases:

  • S phase: DNA replication

  • M phase: chromosome segregation + cell division

G0 phase

• undivided cell can enter G0 phase (quiescent cells: reversible nonproliferating state)

• G0 cells can re-enter cell cycle or become senescent (stable, permanent cell cycle arrest in response to severe stress/damage)

• cell that has reached end stage of development no longer divide (not dividing ≠ biologically inactive)

• terminal differentiation: many specialized cells have little/no normal capacity to re-enter cell cycle

G1 phase

• growth & decision-making

• double mass of proteins & organelles for daughter cells

• time for cells to monitor external environment

  • assess conditions for entry to S phase

  • unfavorable: cell delay progression through G1 or enter G0

• cell still performs normal function

S phase

• DNA replication

G2 phase

• time for cells to monitor internal environment

• preparation: accumulate proteins & resources for chromosome segregation

• coordination: organize machinery to build microtubules of mitotic spindle

• CDK1 activity increases throughout G2 until level needed to initiate mitosis

  • checkpoint signalling restrains CDK1 activity to prevent damaged/incompletely replicated cells from entering mitosis

M phase

• Stages

  • prophase (condense & organize)

  • prometaphase (capture chromosomes)

  • metaphase (align & biorient)

  • anaphase: separate sister chromatids

  • telophase: rebuild nuclei

  • cytokinesis

• first sign: chromosome condensation

• 2 different cytoskeletal machinery assembled: mitotic spindle & contractile ring

Prophase

• chromosomes condense

• mitotic spindles assemble between 2 centrosomes

• condensin helps chromosome organization

• cohesin holds sister chromatids together until anaphase

Prometaphase

• breakdown of nuclear envelope: allows microtubules to access chromosomes

• kinetochores assembled on centromere of each chromosome connects it to microtubules on mitotic spindle

  • attachment is dynamic (unstable)—must be corrected until stable biorientation achieved (one side to left pole, one side to right pole) to enter metaphase

Metaphase

• chromosomes aligned

• each sister kinetochore connects to opposite spindle pole

• kinetochore microtubules (specialized spindle fibers) connect centromere to spindle poles

• visual alignment of chromosomes lined up on metaphase plate does not mean successful segregation (depends on stable biorientation & attachment)

Anaphase

• during onset, regulated cohesion cleavage → allows separation of sister chromatids (immediately become daughter chromosomes)

• chromosomes move by 2 independent & overlapping processes

  • anaphase A

    • daughter chromosomes move toward spindle poles

    • shortening of kinetochore microtubules by depolymerization at two spindle poles

    • forces generated mainly at kinetochores

  • anaphase B

    • poles move farther apart as spindle elongates

    • antiparallel interpolar microtubules from opposite poles generate sliding force to push poles apart

    • motor proteins at poles pull poles apart

Telophase

• daughter chromosomes arrive at spindle poles

• de-condense: chromosomes return to interphase chromatin organization

• reform intact nuclei: nuclear envelope reforms around each chromosome set

• mitotic spindles disassemble

• inactivation of M-phase cyclin-CDK (allows exit from mitosis)

Cytokinesis

• cytoplasm divided into two by contractile ring of actin and myosin II filaments

• creates two daughter cells (each with all essential cell components including organelles)

Organelles during cell cycle

• interphase: ER is continuous with nuclear membrane & organized by microtubules

• M phase:

  • reorganization of microtubules releases the ER

  • ER fragment formation as nuclear envelope breaks down

  • golgi fragments too

  • organelle fragments associate with spindle microtubules via motor proteins → move to daughter cells

Mitotic spindles

• motor proteins slide & pull to organize spindle

  • kinesin: move towards + end

  • dynein: move towards - end

• types of microtubules:

  • kinetochore

  • astral:

    • radiate outward in all directions from centrosomes

    • anchor poles to cell membrane

  • overlap (interpolar)

    • interdigitate at equator of cell

    • responsible for symmetrical bipolar shape of spindle

    • interact with each other & motor proteins (do not attach to chromosomes or kinetochores)

DNA damage & replication checkpoints

• DNA damage/replication stress → checkpoint signaling → CDK restraint

• p53→p21 route

  • damage can stabilize p53

  • p53 induces p21 which inhibits cyclin-CDK complexes & supports arrest

• replication/G2 route

  • ATM/ATR and checkpoint kinases help limit progress

  • regulation of CDK1 can delay mitotic entry

Spindle checkpoint

• regulates entry into anaphase

• unattached kinetochore → checkpoint signaling inhibits APC/C-Cdc20 and arrests cell cycle

• stable attachment permits APC/C-Cdc20 activation

• APC/C marks proteins for degradation

  • control siste chromatid separation: securin degradation → frees separase → separase cleaves cohesin → sister chromatids can separate

  • cyclin degradation → lowers CDK1 activity → promotes exit from mitosis

CDKs

• cyclin dependent kinases

• core enzyme of cell-cycle control system

• phosphorylates specific target proteins, drives cell forward through phases, checkpoints, transitions

• CDK activity oscillates through cycle

Cyclins

• initiate or regulate cell cycle

• cycle of synthesis & degradation

• G1/S Cyclin binds CDK at end of G1 and commits cell to DNA replication

• S-Cyclin binds CDK during S phase to initiate DNA replication

• M-Cyclin binds CDK and promotes mitosis