Cell Bio Exam 3

Golgi and Glycosylation, Cell Signaling I & II, Endocytosis, Phagocytosis & Pinocytosis, Cell Migration, Cell Polarity, Cell Cycle I & II, Apoptosis, Cancer

Golgi structure

stack of flattened sacs

* __cisternae__: individual compartments/sacs, membrane-bound
* __lumen__: inside of compartments
* __cis face__: faces the ER, incoming cargo vesicles
* __trans face__: faces the cell membrane, outgoing cargo vesicles

Golgi roles: protein sorting

“distribution warehouse”

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all vesicles coming from the ER go to the Golgi. The Golgi is where they are sent to their destinations.

Golgi roles: modification of secreted and cell surface proteins

types of modifications

* __N-linked glycosylation__: initiated in ER, present on all secreted proteins
* __O-linked glycosylation__: occurs in Golgi, present in proteoglycans
* __sulfation__: adds a sulfate (SO4)
* __phosphorylation__: adds a phosphate (PO4)

glycosylation

attaching sugar to proteins (different types of simple sugars in a larger structure)

* __O-linked__: attach to hydroxyl groups of Ser and Thr (smaller, less branched)
* __N-linked__: attach to nitrogen groups of Asn (larger, more branched)

glycosylation process: initial glycosylation

begins during **ER import**

* __oligosaccharyl transferase__: transfers the same pre-built sugar to certain **Asn residues** of the protein
* One of the branches on the oligasaccharide has **three glucoses** on the end

glycosylation: processing

N-linked oligosaccharide is modified in the ER and the Golgi

* goes through a series of enzymes, all the way through the Golgi
* **resident glycosylases** are each concentrated in a specific cisterna
* the order of modifications is important, at the end the final patterns all look different

cargo transport: vesicular transport model

**proteins move, glycosylases stay**

* each cisterna is a bag of resident glycosylases


* a vesicle with a cargo protein fuses with the first cisterna and moves through each sequentially

cargo transport: cisternal maturation model

**glycosylases move, proteins stay**

* incoming cargo vesicles fuse together to create a new cisterna
* the cisterna moves through the Golgi and matures
* resident glycosylases move backwards through cisternae
* **experimentally confirmed**

homotypic fusion

Vesicles that leave the ER have **t-snares** and **v-snares**

* **NSF** untwists the v-snare and t-snare
* nearby vesicles fuse together

golgins

**golgins**: tethering proteins on the cisternae

* form a mesh that keeps transport vesicles from floating away
* vesicles can’t “jump ahead”

reasons for glycosylation

* increases solubility
* slows protein degradation (sugar coating)
* determines protein localization
* facilitates protein folding in the ER (N-linked)
* required for protein function

glycosylation and protein folding

correct glycosylation and folding are required to leave the ER

* **chaperone proteins** will remove one of the 3 glucoses when it’s done helping with folding
* when there’s one glucose left, the protein binds calnexin
* **calnexin**: tucks in hydrophobic parts, removes the last glucose

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__misfolding response__

* **glucosyl transferase**: puts a glucose back on if the protein isn’t folded right, sends back to calnexin
* if a mutation keeps the protein from folding correctly, it gets stuck in the ER lumen
* **mannosidase I**: cleaves a mannose
* **EDEM**: sends protein to proteasome

glycosylation and protein function

recognition and signaling


1. signal transduction
2. receptor trafficking
3. ligand secretion
4. signaling (ligand presentation)
5. extracellular matrix formation

stages of cell signaling

1. __Reception__: signal is brought from the outside of a cell to the inside
2. __Transduction__: signal is processed by the cell
3. __Response__: something changes in the cell

types of ligands

__Makeup__**:**

* Chemical
* Protein

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__Source:__

* Environmental
* Manufactured in organism

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__Location:__

* Free floating
* Bound to a neighboring cell

range of ligands

__autocrine__: cells signal to themselves

__juxtacrine__: signal to adjacent cells

__paracrine__: signal across a distance

__endocrine__: signal throughout entire organism

types of receptors

__cell surface receptors__

* bind factors that are **outside cell**, can’t access inside of cell (proteins, peptides)
* **transmembrane** proteins that often act through **dimerization** or **conformational** changes or **both**

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__nuclear receptors__

* bound by **steroid hormones** that pass through cell membrane
* receptor **localizes to nucleus** and initiates gene transcription changes

signal transduction machinery: kinases and phosphatases

__kinases__: phosphorylate

__phosphatases__: de-phosphorylate

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__phosphorylation cascades__

* proteins will phosphorylate then next one in line
* each one phosphorylates several molecules
* **amplification** of signal

signal transduction machinery: small GTPases

__GTPase__: Ras

__cellular function__: bind and activate effector proteins

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*Often these GTPases will have enough GTPase activity to convert GTP → GDP themselves (without a GAP)*

signal transduction machinery: binding proteins

binding can have a positive or negative effect on the target

* __example__: binding protein to target keeps it outside nucleus, phosphorylation of protein causes dissociation and movement of target into nucleus

signal transduction machinery: second messengers

__second messengers__: abundant, small, non-protein molecules

* extracellular ligand was “first”, messenger is “second”
* ex: gases, cAMP, Ca2+

measuring signal transduction activity: FRET

__FRET__: fluorescence resonance energy transfer

* only type of fluorescence that can be used to show that **proteins interact**
* **resonance transfer** only works if molecules are right next to each other

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fluorescence occurs when a protein is ^^excited by light at one wavelength^^, and then %%emits light at a different wavelength%%

* the **light emitted by one protein is absorbed by another** protein, but only if they are close together

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__example__:

* protein X (donor): absorbs purple, emits blue
* protein Y (acceptor): absorbs blue, emits green
* if we see green fluroescence, then proteins must interact
* if we see blue, proteins don’t interact

measuring signal transduction activity: phosphorylation

__PAABD__: binds phosphorylated amino acids

* add donor, acceptor, and PAABD on protein


* if protein gets **phosphorylated, PAABD will bind** to it
* protein **folds in** on itself, bringing donor and acceptor close enough together for a **FRET/interaction signal**

gradual vs binary response

__gradual response__: A → B

* short/simple
* response depends on amount of ligand present
* activity in every cell is increased bit by bit

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__binary response__: A → B → C until enough C for C→ D

* long/complex, amplifying steps
* need enough of C to reach a threshold
* individual cells are activated one at a time

positive feedback loop system

__purpose__: make a long-term change in response to a short signal

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__effects__:

* amplifies the signal
* keeps pathway active even after ligand is removed

negative feedback loop system

__rapid feedback__ (short delay)

* equilibrium is reached at some level below the max

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__slow feedback__ (long delay)

* oscillation between two levels

preventing signaling crosstalk

often, a particular kinase phosphorylated many targets

* need to localize a response, make it specific to its own pathway

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__ways to prevent crosstalk__

* __docking sites__: recruit signal proteins to plasma membrane
* __scaffold proteins__: assemble transduction molecules onto a scaffold, anchor to one location
* __assemble signaling complex on an activated receptor__: binding sites created on receptor when ligand binds

RTK pathway: steps

Pathway that tells cells to divide


1. @@EGF@@: epithelial growth factor, ligand
2. @@EGFR@@: EGF receptor
3. @@EGFR-P@@: phosphorylated receptor
4. Grb2: adapter, mediates between receptor and GEF
5. SOS: Ras GEF
6. Ras: GTPase
7. %%Raf%%: MAP kinase kinase kinase
8. %%Mek%%: MAP kinase kinase
9. %%ERKs%%: MAP kinase
10. %%C-Jun%%: transcription factor, effector

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Steps 1-3: @@ligand and receptor@@

Steps 4-6: adapter and GTPase

Steps 7-9: %%MAP kinase cascade%%

RTK pathway: receptor tyrosine kinases

__transmembrane protein__: extracellular domain and tyrosine kinase domain

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binding of the ligand causes **dimerization** of the receptor

* once the receptor is dimerized, it is **phosphorylated**
* the two receptors **phosphorylate each other (act in trans)** rather than themselves (act in cis)
* **adapter (Grb2) then binds** to receptor

RTK pathway: GTPase and kinase cascade

1. **Grb2** (adapter) recruits **SOS** (Ras-GEF)
2. Ras-GEF activated **Ras** (on membrane)
3. Active Ras binds **Raf** (MAPKKK)
4. Raf (MAPKKK) phosphorylates **Mek** (MAPKK)
5. Mek (MAPKK) phosphorylates **Erk** (MAPK)
6. Erk (MAPK) **phosphorylates** a bunch of other stuff.

RTK pathway: type of pathway

__Binary__:

* amplifying steps: Ras-GEF, kinases

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__Negative feedback__:

* want cell to have enough time before it divides again

JAK-STAT pathway

1. Transmembrane cytokine receptors (with JAK bound) **bind ligand** and **dimerize**.
2. **JAKs phosphorylate** each other.
3. JAKs **phosphorylate the receptor** that they are attached to. This creates a binding site for STAT.
4. **STAT binds** to receptors.
5. JAKs **phosphorylate nearby STATs**.
6. **STATs dissociate** from receptors and **dimerize**.
7. STATs **localize to nucleus** and bind to DNA and other regulatory proteins (affect gene transcription).

Notch signaling

The transmembrane **Notch receptor** binds **Delta**, a membrane-bound ligand on a neighboring cell.

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**Cytoplasmic tail of Notch is cleaved** and moves to the nucleus to interact with nuclear proteins.

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Type of pathway: simple, linear response

* Juxtacrine signaling

G-protein coupled receptors

__G-protein__: trimeric complex of alpha (GTPase), beta, and gamma

A **G-protein-coupled receptor** in the membrane is activated by **ligand binding**.

A cytoplasmic conformational change causes the **G-protein to bind** and the receptor to **act as a GEF, activating G-alpha**.

The **alpha subunit dissociates** from the beta-gamma subunit. These two **pathways diverge**, but both act through **second messengers.**

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__Examples of 2nd messengers__:

* cAMP (bound by G-alpha)
* phospholipase C (bound by G-beta-gamma)
* Ca2+

three pathways for endocytosis

1. Receptor-mediated endocytosis (all cells)
2. Phagocytosis (specialized cells)
3. Pinocytosis (all cells)

receptor-mediated endocytosis: overview

__purposes__:

* bring macronutrients into the cell (too big to cross membrane)
* cell signaling pathways (especially downregulation)

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__two final destinations__:

* send back to cell surface
* degraded in lysosome

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The proteins for this process come from the Golgi (major source), the cell surface, the cytoplasm, and the lysosome.

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**the cell never brings stuff in intact--always breaks it down*

major steps of receptor-mediated endocytosis

1. Vesicle formation
2. Sorting
3. Endocytic Maturation
4. Endolysosome Formation

receptor-mediated endocytosis: vesicle formation

interaction with extracellular receptors to form a clathrin-coated vesicle.

* **PIP2** stimulates endocytosis
* in the presence of PIP2, **AP2** will bind PIP2 and then **cargo receptors**
* AP2 recruits **clathrin** coat proteins (inside of cell)
* structure of clathrin promotes formation of a ball shape
* **dynamin** forms a sprial around “neck” of vesicle
* dynamin contracts through **GTP hydrolysis**, and the vesicle is “pinched” off
* vesicle **uncoats**

phosphatidylinositols

a type of lipid with two fatty acid groups and a **6C ring as a head group**.

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The 6C head group can be **phosphorylated** at every combination of positions 3, 4, and 5.

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Two most important:

* **PIP2**: PI(4,5)P2
* **PIP3**: PI(3,4,5)P3

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**PI3 kinase**: converts PIP2 to PIP3

**PTEN**: converts PIP3 to PIP2

receptor-mediated endocytosis: sorting

vesicles are fused into early endosome

* destination is decided (point of no return)

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__two fates__: recycling or degradation

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__two pathways__:


1. Stimulated: signaling pathways
2. Constitutive: bring in macronutrients

constitutive endocytosis

receptors are continuously endocytosed, whether ligand is bound or not

* receptors release ligand into early endosome (destined for lysosome)
* receptors are recycled to cell surface
* purpose is to bring in macronutrients

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__example__: LDL receptors

stimulated endocytosis

receptors are only endocytosed when bound to ligand

* receptors release ligand into early endosome (destined for lysosome)
* signal is degraded (has done its job)
* receptors are sometimes recycled and sometimes degraded

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__example__: EGF

* __low EGF signal__: receptor dimer is phosphorylated, recycled to cell membrane
* __high EGF signal__: receptor dimer is ubiquitinated, degraded in lysosome

receptor-mediated endocytosis: endocytic maturation

3 major processes


1. multivesicular body formation
2. glycosylation of inner membrane
3. acidifcation of endosome

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product: late endosome

endocytic maturation: multivesicular body

__multivesicular body__

* need to get membrane proteins inside endosome so they can be degraded
* **ESCRT proteins** make uncoated vesicles (push into middle of endosome)
* these vesicles are eventually fully degraded

endocytic maturation: glycosylation and acidification

__glycosylation__

* the endosome membrane needs to be protected from degradation
* inner surface of membrane is glycosylated
* forms a sugar shell coating (**glycocalyx**)
* enzymes to form this come from Golgi

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__acidification__

* **ATPase** proton pumps in the endosome membrane pump **H+** in

receptor-mediated endocytosis: endolysosome

the lysosome fuses with the endosome

* lysosome enzymes come from Golgi
* everything is digested by enzymes
* transporters kick nutrients out to be recycled

Where do the enzymes necessary for endosome maturation and endolysosome formation come from?

__Cell surface__: receptor

__Golgi__: ATPase, glycosylases, lysosome enzymes

__Cytoplasm__: clathrin, AP2, ESCRT proteins

__Lysosome__: degradation enzymes

phagocytosis: purpose

bringing in large particles

* occurs in immune cells
* no coat proteins
* **pseudopods**: membrane extends around to envelop bacteria

phagocytosis: steps

* %%**surface receptors**%% bind %%**bacterial ligands**%% on the outside of the cell
* %%**PI3-kinase is activated**%% on cytoplasmic side of membrane
* PI3-kinase converts PIP2 to %%**PIP3**%%
* %%**actin polymerization**%% is stimulated, which pushes out on the membrane
* membrane grows around the bacteria until it meets itself on the other side (%%**pseudopods fuse**%% together)
* %%**phagosome**%% is formed: membrane-bound compartment inside the cell
* fusion with lyosome forms the %%**phagolysosome**%%

pinocytosis: purpose

“cell drinking” - many pinocytic vesicles line the cell surface

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__membrane balance__

* keep cell from getting bigger and bigger from enterograde vesicles
* exocytosis = endocytosis + pinocytosis

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__caveolin regulation__

* __stretched membrane__: caveolin can’t embed, decrease pinocytosis, allow membrane to grow
* __loose membrane__: caveolin embeds, increase pinocytosis, membrane shrinks

pinocytosis: steps

* %%**caveolin**%%, a coat-like protein embedded in the membrane, induces curvature of the membrane
* %%**dynamin**%% pinches off the “neck” to separate the vesicle using %%**GTP**%%
* late endosome → %%**lysosome**%%

two types of cell movement

1. Flagellar (propellor-like)
2. Amoeboid

overview of cell migration

1. Extension
2. Adhesion
3. Translocation
4. De-adhesion

cell migration: extension

__lamellipodia__: broad extension containing branched actin

* **Rac1** (GTPase) binds to membrane
* activates **Arp 2/3** (**branching actin**)

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__filopedia__: long thin projections of bundled actin

* **Cdc42** (GTPase) binds **PIP3** and recruits **N-WASP**
* N-WASP **unfolds** and forms **90 degree** angle ro membrane
* N-WASP binds **Arp 2/3**
* **actin polymerizes perpendicular** to membrane, making “spikes”

cell migration: adhesion

__retrograde flow__

* **myosin motors** pull growing actin filaments away from the membrane
* when the cell isn’t moving, growth and retrograde movement are in equilibrium

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__focal adhesion formation__

* focal adhesions form to lock actin filament in place (**stops retrograde flow** at leading edge)
* anchor actin to ECM through integrins
* **creation of stress fibers**: actin fibers at front, actin + myosin fibers at back

stress fibers

Complexes of actin and myosin.

Found at back part of cell.

Extend between two focal adhesions.

cell migration: translocation

__contraction of actin fibers__

* **Rho GTPase** activates Rho kinase
* **Rho kinase** activates Myosin light chain kinase
* **MLC kinase** activates myosin light chains
* Causes **contraction**

cell migration: de-adhesion

__release of focal adhesion at back of cell__

* **FAK protein** is phosphorylated
* FAK **phosphorylates adaptor** proteins
* **integrins release** ECM and are endocytosed (recycle to front of cell)
* **actin/myosin** complexes are released

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**no GTPase*

spatial separation of GTPases

__front of cell__: Cdc42, Rac1

__back of cell__: RhoA

**not a GTPase, but FAK is also only active in back*

types of cell polarity

1. __Apicobasal__: top to bottom
2. __Planar__: left to right
3. __Chemotaxis__: front to back

major tools to set up polarity

1. __Extracellular contacts/cytoskeleton__: binding to other cells and ECM can establish ‘sides’ of the cell
2. __Barriers__: if not limited, lipid-bound proteins would move throughout the cell
3. __Directed exocytosis__: controlling destination of proteins maintains polarity
4. __Lipid modification__: control docking/localization of certain proteins
5. __Positive feedback__: reinforcement of localization

apicobasal cell polarity

epithelial sheets of cells

* __lateral side__: cell-cell contacts, tight junctions, cadherin-based adhesions
* __basal__: cell-ECM contacts, integrin-based adhesions
* __apical__: no adhesions

apicobasal cell polarity pathway

1. **tight junctions** form
2. **Par3/6** binds to tight junctions


1. **Par1** binds to cadherin junctions and **restricts Par3/6** to apical region
3. Par3/6 recruits **Crumb**


1. Par1 recruits **Scribbled**
2. Scribbled **restricts Crumb** to apical region
4. The Par3/6, Scribbled and Crumb **complexes** then help designate the **destinations for vesicles** coming from the Golgi


1. positive feedback loop

planar polarity

left-to-right cell polarity

* **proximal** and **distal** sides of cell

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key example is fruit fly wing bristles

* __cell autonomous__: phenotype comes from mutation in own cell
* __non-autonomous__: phenotype comes from outside of the cell
* since mutant **frizzled** cells affect surrounding WT cells, this mutant is **non-autonomous**

planar polarity pathway

1. Transmembrane protein **frizzled** (Frz) recruits **disheveled** (Dsh) and forms a complex
2. Frizzled/disheveled complex binds to transmembrane protein **Van Gogh** (Vng) on neighboring cell
3. Van Gogh recruits **prickle** (Pk)
4. Prickle **inhibits frizzled** at that spot on the membrane
5. Directed **exocytosis** of receptors, etc

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__Result__: Frz and Dsh on one side of cell, Vng and Pk on other

chemotaxis

the cell responds to a gradient of signal (relative concentration), established by spatially separating GTPases

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__Process__:


1. **Chemoattractant receptors** are activated more on one side of the cell.
2. Receptors bind ligand and **recruit PI3k**.
3. PI3k **creates PIP3**. PIP3 concetrations are higher at the front of the cell.
4. **PTEN binds PIP2** in order to create more PIP2. PTEN localizes to back of cell.
5. **PIP3 recruits Rac1** GTPase, which stimulates **actin polymerization** at the front of the cell.
6. **Rac1 inhibits RhoA**, causing RhoA to localize at the back of the cell.
7. **PTEN interacts with FAK** at the back of the cell.

cell cycle overview

flow cytometry and FACS

__flow cytometry__

* label cells with fluorescent dye (label DNA or protein markers)
* cells are passed one by one through a laser that excites fluorescence
* fluorescence is detected by a signal, allowing you to measure proportion of cells above a certain fluorescence threshold

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__FACS: fluorescence activated cell sorting__

* after flow cytometry, cells are passed through a field
* a charge is applied based on fluorescence detection
* cells are separted into two collection cells based on charge

steps of M phase

1. Prophase
2. Prometaphase
3. Metaphase
4. Anaphase
5. Telophase
6. Cytokinesis

prophase overview & centriole duplication

__identified by__:

* chromosomes condense
* centrosomes split and migrate to opposite side

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__centriole duplication__

* S phase - each centriole grows a new partner, these stay together
* M phase - the pairs of centrioles migrate away from each other

prophase: chromatin condensation

helical DNA → (2000 fold packing) interphase chromosomes → (10000 fold packing) prophase chromosomes

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__process:__


1. __histone modification__: phosphorylate Ser
2. __condensins__: help condense chromosomes, loop DNA around


1. come together in disks that pack together
3. __cohesins__: hold chromosomes together

prometaphase overview

__identified by__:

* breakdown of nuclear membrane into little balls of membrane
* microtubules hook up to chromosomes

prometaphase: microtubule attachment

Microtubules (MT) attach to centromeric kinetochore

* **Ndc80 complex**: bridge between MT and kinetochore

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__checking correct orientation__

* growth and catastrophe of MT
* only stabilized if attached correctly (conformational change in kinetochore)

metaphase overview

__identified by__:

* chromosomes line up in middle of cell

formation of mitotic spindle

__types of MT__:

* **astral MT**: grow towards edge of cell, anchor to plasma membrane
* **kinetochore MT**: hooked to chromosomes
* **interpolar MT**: grow towards middle, bound by motor proteins

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__stabilization__:

* if interpolar MT overlap a bunch, motor proteins pull them apart
* if they don’t overlap enough, motor proteins pull them together

anaphase overview

__identified by__:

* chromosomes are pulled apart toward opposite centrosomes

chromosome segregation

__protein complexes__

* **securin-separase complex**: securin is degraded, which frees separase
* **separase** removes cohesin

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__microtubules__

* **astral MT**: catastrophe from + end, shortened to pull centrosomes outward
* **interpolar MT**: no catastrophe, separated by motors
* **kinetochore MT**: catastrophe from + and - end

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__forces pulling on chromosomes__

* depolymerization of MT at + end
* depolymerization of MT at - end

telophase overview

__identified by__:

* chromosomes reach ends of cell
* chromosomes decondense
* nuclear envelope reforms

nuclear envelope re-formation

Little **balls of nucleus** are attracted to the DNA and bind to it. These balls **fuse together** and eventually become one membrane again. The lamin network and interactions with **BAF** are re-established.

cytokinesis overview

__identified by__:

* pinching of plasma membrane
* divison of cytoplasm (rather than nucleus)

cytoplasmic division

cell divides along the metaphase plate

* protein complexes form contractile ring
* **contractile ring**: specialized stress fiber that lines inside of cell made from myosin and actin
* **RhoA** stimulates ring contraction
* **Rac1** at opposite sides extend membrane to pop apart cells

cyclin discovery

forward genetic screen

* create temperature sensitive mutants
* plate on solid media at permissive temperature
* stamp to replica plate at restrictive temperature
* look for dots that are on solid plate but not on replica plate

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cycling of protein expression

* family of proteins that are synthesized and degraded rapidly in in the cell cycle

cyclin-dependent kinases

Cdks (cyclin-dependent kinases)

* target proteins essential for cell cycling (many are transcription factors)
* always present but only activated when bound to cyclin
* cyclin-Cdk complex phosphorylate other proteins to drive DNA replication and mitosis

Cdk regulation

* **Cyclin expression**
* each cyclin-Cdk complex triggers degradation of the previous one
* **Inhibitor binding**
* inhibitor wraps around cyclin-Cdk complex
* **Phosphorylation**
* Cdk is phosphorylated to either activate or inactivate
* **Regulated degradation**
* generally all cyclins & Cdks degraded during anaphase

G1→S transition (starting division)

**E2F** transcription factor is inhibited by **Rb**.

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__Signal for division__:

* **G1/S Cdk-cyclin complex** is activated
* Rb phsophorylated
* Rb released E2F
* E2F activates **S phase genes** (including S cyclin)

G1 checkpoint

__checks for__: no DNA damage

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**ATM** and **ATR** check for DNA damage.

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__If damage__:

* ATM & ATR phosphorylate **p53** (activates)
* p53 activates transcription of **p27**
* p27 inhibits **S Cdk-cyclin** complex

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The cell has a certain amount of time to repair damage or apoptosis occurs. When damage is repaired, p27 released the S Cdk complex.

G2→M transition

__during G2 phase__:

* **M Cdk-cyclin** complex is present
* **Wee1** phosphorylates M Cdk-cyclin to keep it inactive

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__end of G2 phase__:

* **Aurora B** expression activated
* Aurora B activates **Plk1**
* Plk1 phosphorylates **Wee1** (inactivates) and phosphorylates **Cdc25** (activates)
* Cdc25 is a phosphatase that dephosphorylates (activates) **M Cdk-cyclin**

G2/M checkpoint

__checks for__: DNA fully replicated

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**ATM** and **ATR** check for DNA replication (unreplicated DNA looks like damage).

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__If damage__:

* activate **p53**, activate **p27**, inactivate **M Cdk-cyclin**
* ATM and ATR inhibit **Cdc25**, which keeps M Cdk-cyclin from being activated

metaphase→anaphase transition

**APC**: anaphase-promoting complex

* APC is inactive during metaphase
* at end of metaphase, APC binds to **Cdc20** to form active **E3** complex (ubiquitin ligase)
* targets for **ubiquitination**/degradation: securins, cyclins, other cell-cycle proteins

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\*securin degradation frees separase to degrade cohesin, allowing for chromosome segregation in anaphase

spindle checkpoint

__checks for__: proper attachment of all spindles

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Unconnected kinetochores bind proteins to form **spindle assembly checkpoint (SAC) complex**.

* SAC complex binds and sequesters **Cdc20**

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__Once all spindles are attached__:

* Cdc20 is released (activated)

reasons for apoptosis

1. Get rid of unwanted cells during development (sculpting)
2. Maintenance of organ size
3. Cell is infected with virus
4. Cell no longer needed (metamorphosis, immune cells after infection)
5. Too much DNA damage
6. Cell is damaged/stressed out

two mechanisms of cell death

1. __Necrosis__

Cells swell and explode. Cell contents spill out. Frequently leads to inflammation.

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2. __Apoptosis__

Cells shrink and fragment. Apoptotic bodies are phagocytosed. No inflammation.

process of apoptosis

* DNA and organelles are broken down
* Membrane blebbing
* Cell breaks into pieces (apoptotic bodies)
* Phagocytosis of apoptotic bodies

phases of apoptosis

1. __Initiation__


1. __Intrinsic Pathway__: respond to signals inside cell
2. __Extrinsic Pathway__: respond to signals outside cell
2. __Execution__


1. both initiation pathways converge here

caspases

signaling proteins that act through **cleavage methods**

* cleavage is irreversible
* don’t want to accidentally activate

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__initiator caspases__:

* activated by apoptotic signal
* dimerize
* cleave executioner caspases to activate them

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__executioner caspases__:

* activated when cleaved
* multiple targets-- cause apoptosis

extrinsic apoptotic pathway

caused when a signal is received from another cell, like an immune response to a virus infection.

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__Process__:

* **killer T-cells** check proteins displayed on cell surface
* when a foreign protein is detected, T-cells secrete **Fas ligand**
* Fas ligand binds to **Fas death receptor** on targeted cell
* receptor trimerizes to form **DISC complex**
* **FADD adaptor** binds to death domain
* **initiator caspases** bind to adaptor
* initiator caspases are cleaved and dimerize
* initiator caspases cleave and activate **executioner caspases**

intrinsic apoptotic pathway

caused by an internal signal, like DNA damage.

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__Process__:

* **anti-apoptotic proteins (Bcl-2)** in the cytoplasm bind and inactivate **pro-apoptotic proteins (Bax, Bad)**
* **p53** causes Bcl-2 to release pro-apoptotic proteins
* pro-apoptotic proteins embed in mitochondrial membrane and form a pore
* **Cyt C** flows out of intermembrane space into cytoplasm
* Cyt C binds **Apaf-1** to form CARD complex
* 7 **CARD complexes** form apoptosome
* **apopotosome** recruits and activates initiator caspase

executioner caspases

multiple targets

* ==downregulate adhesion proteins==
* anoikis: extrusion of apoptotic cells from epithelial tissues
* %%activate DNA nucleases%%
* caspase cleaves iCAD, which releases CAD endonuclease from inactive iCAD-CAD complex
* %%activate actin regulators%% (break down actin)
* membrane blebbing
* ==inactivate lamins== (nuclear breakdown)
* %%activate and%% ==inactivate flipases==
* inactivate flipases that maintain lipid content
* activate flipases that scramble lipids
* membrane leaflets are mixed up and look foreign

6 hallmarks of cancer

1. Cell growth and division without the proper signals to do so
2. Continuous growth and division even when there are signals telling them to stop
3. Avoidance of programmed cell death
4. Limitless number of cell divisions
5. Promoting blood vessel construction
6. Invasion of tissue and formation of metastases

types of cancer

* __carcinoma__: most common, cancers derived from epithelial cells
* __sarcoma__: cancers derived from connective tissues
* __lymphoma & leukemia__: blood cancers
* __germ cell tumors__: rare, arise from pluripotent cells (usually testicle or ovary)
* __blastoma__: derived from immature precursor cells or embryonic tissue