no flowchart review

Ethical Issues from Epo Doping?

• unfair advantage due to change in body’s abilities

• It’s harmful to the athlete bc it inc BP to strain heart

• Medecine is to be used for healing over personal gain

Parameters to consider when drug development to block a signal pathway

• If you block at receptor, other downstream pathways may be disrupted that aren’t your target

  • Ex. cAMP is a general secondary messanger

• Must consider:

  • Specificity to tissue

  • Specificity to pathway

  • Minimizing side-effects on normal cells

Beta-blockers

• inhibit B-agrenergic receptors to reduce heart rate and BP

• Used to treat hypertension, arrhythmias, anxiety

• Has side-effects though

Taxol Function

• Chemotherapy drug from pacific yew tree

• Stabilizes microtubules to prevent depolymerization during mitosis

• Arrests cell is G2/M phase and triggers apoptosis

Why do cancer drugs target microtubules rather than actin?

• Cancer cells exhibit rapid division

• Microtubules form the mitotic spindle essential in mitosis

• Actin is more for shape and mobility

• Targetting actin would harm all cells bc it plays improtant role there

• Targetting microtubules is more to cancer bc it’s more sucesptible to it since it exhibits over proliferation

Why does cell adhesion matter in cell biology

• cell-cell communciation to maintain tissue architecture

• Immune Repsonse as immune cells must navigate and reach sites of infection

• Pathogen strategies as they exploit adhesion to invade hosts

• Therapeutic implications as understanding adhesion helps us design targeted therapies

UPEC

• Causes UTIs by adhering to host epithelium

• They use pili and adhesins to do this

• Colonize / invade urethra and bladder

• They repress the immune system there

• Form biofilm and damage the epithelial

• Can cause sepsis when it gets to major bloodstream

UPEC adhesion for UTI (Fim H)

• Adhesion needed so they’re not swept away from the host

• They use lectins to do so

• These lectins are on the tip of the pili and can interact with mannose sugars on surface of urinary tract epithelial cells

• Fim H is the lectin region that targets mannose

• Fim H is necessary for this adhesion

UTI Treatment challenges

• Treated by antibiotics

• Challenges: Antibiotic resistance and perturbance of beneficial gut microbiome

Blocking UPEC adhesion to prevent UTI

• Intriduction of competitive inhibitors

• They bind to the lectin regions so they cannot adhere to cell surface

• FimH binding domain can be used to design inhibitors with excellent potency

FimH: Large scale screens

• Determines which inhibitors may work best to inhibit UPEC adhesions

• Incubate UPEC with inhibitors

• Pre-coat wells with mannose

• After incubation, see which inhibitor doesn’t allow UPEC cell binding best

Cranberries in UTI Prevention

• They have proanthocyanidins that are bound by FimH

• UPEC adherence thus decreases with it

Limitations with Inhibiting FimH

• Bacteria have manyyy pili making it difficult to block them all

• They also express many adhesino molecules so FimH alone may not be enough

• So, maybe we can develope an array of inhibitos as a therapy

Similarities and Differences with bact/WBC adhesion

• Sim:

  • Similar movement to target tissue

  • Use adhesion to get places

• Diff:

  • Integrin/Selectin for WBC while bact uses lectin to target mannos

  • Bact aggregate to damage while WBC go in to fight

Structure and Function of Kinesin Superfamily

• Kinesin ½ bind cargo

• Kinesin 5

  • 4 ATP binding head chains

  • Allows sliding bc it walks along 2 at once

• Kinesin 13

  • No motor activity

  • Helps in disassembly

MTOC

• centrosome in regular cell

• Located near nucleas and nucleates MTs

• Becomes spindle poles during mitosis

Spindle Assembly

• Each centrosome has a pair of centrioles

• MTs nucleate in PCM which have y-TuRC where they polymerize

Centrosome duplication

• occurs at G1/S when chromosomes are duplicating

• G1/S phase CDKs initiate this duplication

• In G2, daughter centriole growth is complete

Centrosome Splitting

• M phase CDK activates this

• Both MTOC nucleate and polymerize MTs

• The two centrioles are pushed to opposite sides of nucleus and are now spindle poles

• This happens during prophase before NEB

• Mediated by Kinesin 5

  • Walks antiparallel (+) on two MTs allowing them to slide past

MT Types

1. Astral: Project and link to cell cortex

2. Kinetochore: Connect to chromosomes

3. Polar: Project toward cell center and overlap to help push spindle poles apart

How do MTs seperate sis chromatids during mitosis

• Monopolar attachment of one sister chromatid

• Bipolar attachment by 2 MTs

• This tension aligns them at centre

• MTs interact with the kinetochore protein complex of each sister chromatid

• All chromosomes must achieve bipolar attachment to enter anaphase (SAC)

Anaphase A

• Kinetochore microtubule shortening

• Chromosomes are pulled poleward

• Shortening of kinetochore MT pull them towards the spindle pole to seperate sister chromatids

Anaphase B

• Seperation of spindle poles

• Sliding force egenrated b/w polar MTs to push them apart

• This acts on the poles to move them apart

• Also MTs grow at the (+) to keep kinesin 5 to have the overlap needed to generate this sliding force

Cancer

• Loss of normal control of cell proliferation

  • Large virable nuclei

  • Variation in size/shape

  • loss of normal specialized features

  • Large # of dividing cells

• It’s mamed for which cell type it begins in

how are tumors different from normal cells

• Changes in genomes (many types of mutations)

• Tumours have different cell types that interact with their env to obtain max growth advanatge

• Metastatic tumours have migrstory properties

causes fo cancer

• Many small changes allowing the cell to be best-suited for uncontrolled growth

• Acquired mutations are most common, from risk factors

  • Tobacco

  • UV

  • Toxins

  • Age

• Germline mutations

  • Mutations in sperm/egg

  • Passes from parent to child so inherited cancer

Mutations for cancer

• cancer is from many mutations over a lifetime

• 1. tumor suppressor genes

  • Moniter cell division

  • repairs mismatched DNA

  • Ex. BRCA1/2

  • Ex. p53 or TP53

• Most p53 gene mutations are acquired

• 2. Oncogenes

  • Turn healthy cells cancerous when mutated

  • Ex. HER2

  • Ex. RAS

• 3. DNA repair genes

  • Fix mistakes when DNA is copied

  • Mutations here is a proble

  • Ex. BRCA1/2

Deregulation of cell cycle in Cancer maintenance

• Overexpression of proto-oncogenes

• LOF mutations in tumor suppressors

• LOF in p53

• Mutations in genome-maintenance genes

Cancer treatments

1. Radiation therapy: High dose of rad to kill and stop spread

2. Chemotherapy: Chemical killer to weaken cells

3. Surgery: Removing tumor

4. Immunotherapy

Cancer drug development

• it’s becoming more prevelant in younger people too so new promising therapies may not be so effiicent

Drug Resistance

• Cell pumps out toxins so it’ll pump out this toxin and over-express the mechanism for it for next time

• Repopulation (they’ll come back after dying)

• Drug distribution (one exposure to it can cause it to mutate to not be vulnerable)

p53

• tumour suppressor and TF regulating cell devision to prevent tumors

• Stops cells with damaged DNA from dividing and signals apoptosis

• Or it arrests the cells if not too severe damage

p53 Mutation

• Cell division needs balance between proto-oncogenes and tumor supressors

• Disrupts mitotic checkpoints so cancer can divide indefinitely

• Withou p53, there’s no halting of cell cycle or signal for apoptosis

  • no DNA repair

p53 and cancer

• p53 is mutatued or deleted in cancer and the pathway for it is disrupted

• Allows cancer to evade checkpoints and apoptosis

• It’s challenging to use the pathway for therapy but different strats are effective

p53 and cancer therapeutic challenges

• targeting mutant p53 is identifying a binding site for it

• It’s also in the nucleus mainly which is hard to access esp in cancer cells

• There are many types of mutations which can be present in p53 for different molecules of it

p53 and cancer promising solution

• Y220C mutant p53 PC14586

• PC selectively binds to p53 Y220C and restores it

• It creates a binding pocket for small inhibitors

Why does regulated cell death occur?

• Normal part of cell life cycle

• It’s an eqm where you gain and lose as a constant process

• Helps in preventing cancer i guess by not having too little cell loss

Apoptosis in embryo hand/feet development

• Even in early development, apoptosis takes place 

• In week 6, skin forms webbing between the digits 

• By week 11, the webbing disappears due to apoptosis

Proof of Apoptosis in Embryo Webbing (Mouse Paw)

• Mouse paw embryo stained with a dye to detect apoptosis

• Shown as yellow dots seen mostly where the webbing is

• As webbing disappears, the bright spots do too

• This is done through TUNEL assay

TUNEL Assay in Apoptosis Detection

• Takes advantage of the nicks present in apoptosis cell’s DNA

• dUTP can be incorporated into the nicks by enzymes

  • Enzyme: Terminal Deoxynucleotidyl Transferase

• Flourescently-labelled dUPT can specifically detect cells in apoptosis

Apoptosis in Frog Metamorphosis

• Tadpoles undergo metamorphosis to become a frog

• The tail disappears as the cells are induced to undergo apoptosis 

• This is stimulated by the increase of the thyroid hormone in blood

Apoptosis in Human Nervous System Development

• Half of the cells originally produced are required in normal brain development 

• The other half undergo apoptosis 

  • Cells that haven’t achieved synaptic connections

  • Cells with faulty connections

  • Cells not having made contact with a target cell

• Matches the number of nerve cells with the number of target cells

Inappropriate Cell Death Diseases

• Alzheimers: Neurons in hippocampus and cerebral cortex die

• Huntingtons: Neurons in striatum die 

• Parkinsons: Dopamine neurons in substantia nigra die

• Duchenne Muscular Dystrophy: Muscle cells die

What are the 2 ways in which cells die

1. Necrosis 

2. Apoptosis 

Necrosis

• Cell death through damage to exterior 

• Cells swell and release contents to surrounding tissue 

• Can lead to infection

Apoptosis

• Programmed cell death that is regulated

• Cells suicide in response to stress/damage or as a part of normal development 

• The debris isn’t released to damage cells nearby

• The debris is contained and recycled 

Apoptotic Pathway

• Cell Execution: Kill the cell

• Engulfment: Get rid of the body 

• Clearance: Destroying the evidence

Ultrastructural Features of Apoptosis (7)

• Chromatin compacts and condenses 

• Nuclear envelope breaks down

• Nucleus contents are fragmented and the DNA / proteins are degraded 

• Cytoplasm undergoes condensation as cellular components aggregate 

• Mitochondria is permeabilized and released into the cytosol 

• Cell membrane moves and changes shape to create blebs (protrusions)

• Cell fragments create compartments with debris which will be phagocytized and recycled

C. elegans Apoptosis Model

• They’re studied very much in detail 

• 947 somatic cells have been identified in the adult worm

• The lineage of them all is traced to a single cell undergoing rounds of division 

• 131 cells undergo apoptosis 

Apoptosis Genes Identified by C. elegans model

• Done by assay for identifying mutations in genes 

• These genes are called “cell death genes” (ceds)

• Mutation in ced-1: Allows apoptosis but not the associated phagocytosis 

• Mutation in ced-3: No apoptosis observed 

• Four essential genes:

  • ced-3

  • ced-4

  • ced-9

  • egl-1

Mammalian Apoptotic Pathway

• EGL-1 Homologs: Bid and Bim

• CED-9 Homolog: Bcl-2

  • Bcl-2 controls Bak and Bax

• CED-4/3 form a complex called the caspase holoenzyme 

  • Protease targeting many different proteins for degradation

• CED-3/4 mutations prevent death

• ced-9 mutations make all cells die

  • Inhibits activation of caspase holoenzyme

  • Inhibits apoptosis in this was

  • EGL-1 signals apoptosis by inhibiting CED-9

Caspase Holoenzyme

• Apoptosome in mammalian cell

• Contains direct homologues of C. elegans proteins

• Apaf 1 = CED-4

• Caspase-9 = CED-3

• Protease activity of caspase holoenzyme leads to protein degradation and cell death

Activation of Caspase Holoenzyme (C. elegans)

• In C. elegans

• CED-9 inhibits apoptosis by binding to CED-4 dimers

  • Keeps them inactive 

• EGL-1 binding to CED-9 releases CED-4

• CED-4 then join with CED-3 to form caspase holoenzyme 

• This leads to degradation of cytosolic and nuclear proteins 

Mammalian CED-9 homologue

• It’s Bcl-2 

• Normally anchored to outer membrane of mitochondria 

• Alters permeability of it 

  • It maintains low permeability when present 

  • When inactive, it forms pores associated with apoptosis 

Bad: Apoptosis Signaling Pathway with Cytochrome C.

• Mammalian cell apoptotic signal is called Bad

• It’s inactive while phosphorylated and bound to 14-3-3 

  • 14-3-3 is a cytosolic adaptor protein

• Signalling pathways allow dephosphorylation of Bad

• It then releases from 14-3-3

• It then binds to Bcl-2 on mitochondria

• This activated Bcl-2 to allow for Bax to be activated 

• Bax aggregated into clusters in the membrane to make pores 

• Pores increase membrane permeability 

• Allows release of mitochondrial proteins into cytosol

• This includes cytochrome C which is essential in forming mammalian apoptosome

Trophic Factors in Apoptosis Prevention

• Trophic factors prevent apoptosis to keep the cell alive

• They initiate a kinase cascade leading to phosphorylation of the Bad protein 

• When trophic factors are removed, Bad can be dephosphorylated 

Dictyostelium discoideum slime mold

• Eukaryote

• Transitions from a unicellular amoeba to

  • multicellular slug

  • fruiting body

• Aggregated amoeba form a slug

• They then differentiate into 2 cell types

  • prestalk

  • prespore

• The anterior end of the slug forms the stalk

• Posterior end will form the spores of the fruiting body

Dictyostelium Vegetative Growth Phase

• They feed on bacteria 

• when food is abundant, they divide by mitosis 

• This is vegetative growth phase 

Dictyostelium aggregation

• induced by starvation

• happens in response to cAMP produced by starved cells

• Aggregation forms the slug which moves to find suitable env

Dictyostelium in Suitable Env 

• When it finds a nutrient-rich env, it stops and begins to differentiate 

• Anterior cells form the stalk

• Posterior cells form the fruiting body 

• The fruiting body contains spores with a hard cell wall 

• The spores will eventually germinate to form new single-celled amoebae 

Dictyostelium receptor for cAMP + response

• Transmembrane protein: G-protein coupled receptor (GCPR)

• cAMP binds extracellularly to activate the receptor 

• Cells reorganize their intracellular actin cytoskeleton 

• This allows them to move towards the signal source 

what enables the movement of Dictyostelium towards cAMP source

• Dynamic filopodia extending outwards

• Signaling initiated actin reorganization, including 

  • nucleation

  • polymerization

  • depolymerization

• All of this allows movement to occur

Disctyostelium movement with clathrin heavy chain mutation

• Means cells can’t form vesicles needed for protein transport to cell membrane 

• cAMP is detected by transmembrane GPCR proteins 

• In the absence of clathrin, GPCR isn’t transported to cell surface 

• The cell is then unable to respond to the signal

• There is no net movement towards the signal source

Neutrophils

• WBC in our bodies 

• Can respond to signals made by bacteria invading our bodies 

• it’s irregularly shaped and is able to crawl to follow bacteria around RBCs

Signals enabling neutrophils to follow bacteria

• Bacteria produced a protein signal containing

  • methionine

  • leucine

  • phenylalanine

• Neutrophils have a receptor on the surface able to recognize this fMLP peptide

  • It’s a GPCR recetor

Signaling Definition

• Signaling is the transmission of information from one cell to another that induces a change in behavior.

• Signals are only useful if there’s a response to it

Principles of signal transduction pathway (STP)

• Signaling cells produces and releases signaling molecules

• Target cells have a receptor to bind to the signal 

• This activates the receptor to make a cascade of event

• This cascade will interpret and transduce the signal to cause change in behaviour

  • Transcription 

  • Cell movement / growth / differentiation 

• Many cells may be exposed to the signal, but only the target ones will have receptors to respond

Specificity of signal-receptor interactions in signaling

• They only bind if they have high molecular complementarity

• It must be specific and high-affinity 

• Allows the interacting surfaces to come closer through essential amino acid residues being present 

• Induces conformational change in intracellular domain of receptor 

• This activates the STP to lead to a response

The 2 levels of signal response specificity

1. Specificity of signal for binding to receptor 

2. Specificity if intracellular response mediated by STP

   1. Same signal can activate different intracellular proteins of different cells 

Fast Cellular Response 

• Extracellular signal binds to membrane-associated receptor 

• Activated cytosolic enzyme through a modification (ex. methylation)

• Fast response bc cell can quickly respond by activating an already present protein in response 

Slow Cellular Response

• Binding of signal causes change in protein levels within cell

• A soluble receptor is within the cytosol

• The signal passes through the membrane to bind to it

• Upon activation, the receptor is transported into the nucleas

• It can directly/indirectly act as a transcriptional activator to produce mRNAs

• This can increase protein levels within the cell

• This is a slow response as it depends on many steps that take time

Measuring the Signaling (Enzyme Kinetics like Graph)

• Affinity here measured similar to protein-ligand (pink)

  • 100% means all the receptors are filled

  • x axis: signal conc

  • y axis: fraction of bound receptors

  • Kd: Dissociation constant 

    • [signal] required to have half max binding 

    • represents receptor-signal affinity 

• Measure of [signal] required to produce response (blue)

  • x axis: [signal]

  • y axis: fraction of cells responding 

  • Max response can be measured, half can be calculated 

• [signal] needed to achieve half of response is less than to fill half of the receptors

  • Means signal amplification takes place

Endocrine (Secreted) Signaling

• Signals are released into circulatory system

• Cells throughout the body are exposed 

• Only cells with target receptors can respond

• different cells of different tissues can respond at the same time 

• Common ex: Secreted Hormones 

Paracrine Signaling

• Secreted signals are released into extracellular space

• They can diffuse into neighbouring cells 

• Signal and target cells are close

• Common Ex. Growth factors and NTs

Proximal Signaling 

• Signaling and target cells are in direct contact

• Signal and receptor proteins may be transmembrane proteins on different cells

• So the interaction requires the cells to be attached by adhesion done by integral membrane proteins

Cell Signaling by Cytosolic Messengers (Plasmodesmata / Gap Junctions)

• Ex. In plants (plasmodesmata) and animals (gap junctions)

• They have junctions between cells spanning the cell wall/membrance

• This connects cytoplasm between the cells allowing messengers to move quickly

Autocrine Signaling

• Cell communicating with itself 

• Signaling and target cell is the same 

• Cell produced secreted signal, and also carries receptors for it 

• Ex. Growth factors produced to induce cell division 

Classification of Cell-Surface Receptors

• There are 7 total

• We focus on 3 types 

  • Cytokine receptors 

  • Receptor-tyrosine kinases (RTKs) 

  • G-protein coupled receptors (GPCRs) 

Extravasation

• The movement of WBC from blood stream to surrounding tissue

• 5-step process initiated by a signal created by infection

Why are transient (temporary) cell adhesions important, and when do they occur?

• Not all cell adhesions are permanent — some are temporary to allow movement.

• Transient adhesions are essential for:

  • Cell migration across extracellular surfaces.

  • Cell movement during embryogenesis.

• These connections form and break repeatedly, enabling cells to travel where needed.

How do leukocytes use transient adhesion during an immune response?

• Leukocytes must exit blood vessels to reach sites of infection or injury.

• This extravasation relies on a sequence of temporary adhesive interactions with endothelial cells

• Normally, adhesion between endothelial cells prevents blood leakage

• During an immune response, leukocytes temporarily attach and cross the vessel wall to enter tissues.

What are the 3 families of WBCs / Leukocytes

1. Granulocytes: Neutrophils 

2. Monocytes: Macrophages

3. Lymphocytes: T and B cells 

Granulocytes

• Target pathogens 

• Include neutrophils, eosinophils, and basophils 

Neutrophils

• Most common granulocyte

• Primarily targets bacteria infections 

• One of the first cells to respond to trauma 

• Capable of extravasation

Monocytes 

• They differentiate into microphages 

• They engulf invading bacteria or dead cells through phagocytosis

• Capable of extravasation

Lymphocytes

• Include NK (natural killer) cells

  • Lyse virally infected cells and tumour cells 

• Include T and B cells 

  • Produce antibodies as immune response 

• Can undergo extravasation

What are the five steps of extravasation

1. capture

2. rolling

3. slow-rolling 

4. firm adhesion 

5. transmigration

Extravasation: Step 1

• Capture (Using Neutrophil Ex) 

• This is the transient association between the neutrophil and the apical surface of endothelial cell 

• They’re still being pushed by bloodflow but slower 

• The cells roll along the surface of endothelial cells

Extravasation: Step 2 / 3

• Rolling / Slow Rolling

• Since the transient associations are slowing the neutrophil, it rolls along the surface

• The rate slows down as # of associations increase

• This leads to firm adhesion

Extravasation: Step 4

• Firm adhesion 

• Occurs with stronger attachment of neutrophil with endothelial cells 

• This is accompanied by changes allowing the WBC to break connections b/w endothelial cells 

• This allows migration along the cell surface to outside the blood vessel 

Extravasation: Step 5

• Transmigration

• The seperation of endothelial cells allow the neutrophil to migrate out of the blood vessel

• Causes swelling as transmigration occurs

Extravasation Capture Mechanism

• Cytokines (e.g., TNF-α) are released at the infection site

• They signal endothelial cells of blood vessels.

• This signal (received at the basal surface) triggers endothelial cells to move P-selectins from secretory vesicles to their apical surface.

• P-selectins on the endothelial surface then bind to selectin-specific glycoprotein ligands on neutrophils

• This captures them from the bloodstream and initiates the immune response.

Extravasation Rolling Mechanism

• Adhesion of neutrophil to endothelial cells slow movement 

• Eventually, they start rolling along the walls

• This involves them being pushed over the surface while establishing and losing transient connections

Extravasation Slow-Rolling Mechanism

• Density of selectins on endothelial cells inc closer to site of infection 

  • Many endothelial cells are displayed P and E selectin here 

• The inc associations between selectins and the ligands on neutrophils flows their movement 

• They are no undergoing slow-rolling 

Extravasation Firm Adhesion Mechanism

• Slow rolling lets new interactions form between neutrophils and endothelial cells.

• PAF (platelet activating factor) on endothelial cells binds to the PAF receptor on neutrophils (a

  • Ex. receptors CXCR1 and CXCR2

• This interaction occurs only during slow rolling and activates a signal transduction pathway inside the neutrophil.

• The signal activates integrin adhesion molecules on the neutrophil, enabling them to bind ICAMs on endothelial cells.

• This binding slows the neutrophil further, leading to firm adhesion (tight binding) to the vessel wall.

Integrin Protein Structure (Extravasation Firm Adhesion)

• Inactive integrin (dimeric) has its propeller and β-A domains folded down, preventing ligand binding.

• PAF signaling triggers a conformational change, activating the integrin so it can bind ICAMs on endothelial cells.

• Integrin–ICAM binding is much stronger than selectin interactions, resulting in firm adhesion of the neutrophil.

• Activation also initiates actin cytoskeleton reorganization, preparing the neutrophil for cell migration out of the blood vessel.

Extravasation Transmigration Mechanism

• The neutrophil has stopped at the site of infection

• It can migrate b/w the endothelial cells 

• The connections b/w them are broken by enzymes produced by transmigrating neutrophil

Progressive Activation of Extravasation

• Selectins are activated first

  • Mediates capture, rolling, and slow-rolling 

• Signalling pathways activate integrins 

  • Mediates firm adhesion 

  • Allows transmigration

H.V. Wilson Sponge Experiment 

• First demonstrated the ability of cells to recognize and adhere to one another 

• Used the cells of 2 sponge species 

• Their indiv cells were seperated using a fine mesh 

• The cells were then mixed together 

• Overtime, the cells from the same species were able to recognize and associate back together 

  • Cells from diff species didn’t associate 

Johannes Holtfreter: Frog Embryo Experiment

• Showed cell recognition and adhesion using frog embryos

• Took cells from 2 different developmental germ layers and seperated indiv cells 

• Similar tissue recognized eachother and associated

• The associations mimicked original embryo organization