Bis 104 Unit 3

Counting M Phase Culture cells

Counting M Phase Culture cells

Cultured cells have doubling times of ~20 hrs; if 5% of cells are in M phase it takes 1hr to completely

cdc mutants in yeast

o Arrested during the cell cycle (-): elongated cells with undivided nuclei
o Advanced into M phase too early (D): short cells (the wee phenotype)
o Cloning of cdc genes (+): complementation of loss‐of‐function mutations

What part of cell drives oocyte into m phase

The M‐phase cytoplasm contained a factor that can drive oocyte into M phase

MPF oscillation

increases when cells entering meiosis/mitosis and falls to 0 toward the end of mitosis

M-CDK + M-cyclin

cyclins rise during M phase and drop after mitosis

MPFs

Cdk → also known as Cdc2 protein

Cyclin → B-type; acts as targeting / activating factor for kinase activity

both are needed for activation, but different cyclins are produced to acitvate cdk at different phases

maturation promoting factor — brings cells into m phase

cyclin‐binding & phosphorylation

Activation of Cdk + postiive feedback loop

Ubiquitylation

addition of ubiquitin side chain tothe cyclin protein by three consecutive enzymatic reactions by E1, E2, and E3 enzymes

polyubidquitin side chain = sent for destruction

Degradation of Cyclin to enter anaphase

E3 enzymes ( APC/C and SCF ) specify the target protein to be ubiquitylated!

Degradation of CKI to initiate S phase

E3 enzymes ( APC/C and SCF ) specify the target protein to be ubiquitylated!

APC/Cyclosome (APC/C) and destruction of cohesin complex

Checkpoint mechanisms three components

Sensory: detects chromosome abnormalities

Signaling: transmits information of abnormality

Effector: capable of inhibiting the cell cycle machinery so that it is
halted

Activation of DNA damage checkpoint leads to the expression of the
CKI p21

Activation of spindle assembly checkpoint (SAC) leads to mitotic arrest

o Unattached chromosomes block sister chromatid
separation!
o Anaphase would not start until ALL kinetochores are
captured by microtubules in the spindle.

2 Stages of Anaphase

A. sister chromatid / chromosome separation -→ shortening kinetochore

B. spindle elongation = more pole separation + central spindle elongation

Cleavage by the contractile ring made of Actin microfilaments and Myosin II

o Myosin II walking along actin filaments toward (+)

o Actomyosin ring contracts

o Generates force to cleave cell

phragmoplast

microtubules based array that guides vesicles to drive the formation of cell plate

Asymmetrical call division

Ex. Neuroblast

Stem Cell Division

Apoptosis v Necrosis

Apoptotic cells die neatly, cell wall is destroyed and cell parts are recycled

Phosphatidylserine (PS) - Apoptosis

Faces inner leaflet until apoptosis when it’s displayed = corpse marker

Caspases definition

Targets to cause cell death → kills proteins that aid in proliferation

evolutionarily conserved

Caspase activation

Bak / Bax

Releases cytochrome C from mitochondria = apoptosis signal

Prevention and Induction of mitochondrial outer membrane

permeabilization (MOMP)

Bcl2 = anti-apoptosis

How to prevent apoptosis

Assembly of the apoptosome

• Cyt-C activates adaptor proteins → Apaf1

• Activated Apaf1 assembled into apoptosome with the CARD domain in middle

• Recruitment of procaspase

• Caspase activation at the apoptosome

• Activation of executioner caspases

Apoptosis Summary

Integrin dimer

connects to dimerized fibronectin -→ recognizes rgd

Cell-cell junctions in animal cells

as you get closer to basal, gaps get less dense

• Tight junction: ~0

• Gap junction: 2‐4 nm

• Adhesive junctions: 20‐35 nm

Gap junction

Connexon on one cell pairs w/ connexon on the other cell -→ depends on what’s needed : more connexon = more resources needed

allow molecules <1,000 Daltons to pass (ex ion, atp, glucose, amino acids)

Cadherins

Look for identical cadherins

Ca triggers cadherin recognition - no Ca = no association

Plasmodesmata

vesicle channel to transport nutrients

Cancer Cells with Age

Higher chance of getting cancer as you age

How cells turn into tumors

Mutagen v Carcinogen

Stronger mutagenic activities → stronger carcinogens

Ames test

Identifies potential carcinogens

Genetic disorders associated with cancers

Overactivity - overactive oncogene

Underactivity - underactive suppressor gene

Tel-PDGFR

Rb

Negatively regulates cell cycle progression

Mutations in oncogenes v suppressor genes

3 Major pathways contributing to tumorigenesis

All cancers converge using 3 pathways

1. Rb malfunction - cells constantly enter cycle

2. p53 - reduced tolerance to stress + damage

3. Ras - Signaling cascade

Gleevec

Blocks Bcr-Abl kinase activity

How does HPV Work?

HPV has gone past point of no return when viral gene has infected hose genome

Conversion of proto‐oncogenes to oncogenes

Overproduction of critical genes

iPS

Taken from a patient (already stomatic) that are reprogrammed back into embryonic stem cells

Checkpoint Graph

Flow Cytometry Graphs