Bio 232 lab quiz

Week 2

  • Defective efferocytosis
    • Macrophages (which are scavenger cells) do not eat foam cells after apoptosis leading to secondary necrosis and inflammation
    • M2 phenotype have higher rates of efferocytosis
    • To create foam cells, macrophages can be Ac-LDL treated but this is tedious and expensive.
  • Steps of efficient apoptotic cell clearance
    • Find me signals, eat me signals recognized by phagocytic receptor
    • This lab will focus on an eat me signal called PtdSer found on the bilayer of apoptotic cell
      • Phosphatidylserine flips after eat me signal
    • TAM Mertk scavenger receptor on the surface of macrophages recognize the eat me signals on apoptotic cell
      • Usually in the cytosol side of plasma membrane
      • Flips outside to the extracellular space during apoptosis
    • Briding molecule called Gas6 increases affinity between eat me signal and scavenger receptor on phagocytic cell
    • Cytoskeleton is rearranged to engulf the apoptotic cell
  • Apoptosis
    • Programmed cell death
    • Functions for embryonic development and balancing of cell death/division
    • Cells basically collapse, DNA breaks, cell membrane breaks, shrivels up/condenses, nuclear envelope breaks
    • Proteases (aka caspases) activate from apoptotic signals and initiate apoptosis
    • Amplification cascade results in apoptosis
    • The apoptotic signal can be external or internal
    • This cascade amplifies the signal to start the cleavage of key proteins needed for cells to live
  • Rac1 vs RhoA
    • Rac1 enhances
    • We want increased Rac1 activity to make extensions to engulf apoptotic cells
    • Rac1 is the final signal in macrophages that causes phagocytic cup formation
    • Triggers anti-inflammatory cytokine secretion
    • Results in macrophage moving!
    • RhoA inhibits efferocytosis
    • Triggers inflammation and low efferocytosis
  • Actin related proteins
    • ARP adds itself to the positive end of actin
    • Actin can create protrusions cause contractions that cause a cell to move
  • Week 2 experiment goals:
    • Show that macrophages can eat apoptotic cells
    • Induce apoptosis using UV light
    • Ways to kill cells: lack of nucleotides, UV radiation, ionizing radiation, oncogene signaling (cancer), hypoxia, blockage of transcription
      • These can cause cell cycle arrest, DNA repair, block of angiogenesis, or APOPTOSIS (our target)
    • UV can cause DNA damage which  
    • Effector Resolvin D1 can increase rate of phagocytosis
  • Measurement
    • Fluorescence only stains apoptotic cells because the membrane is permeable to the dye
    • 10-40-80 minute intervals
    • The process will start by binding (scavenger receptors recognizing the PtdSer / eat-me-signal), then start of degradation through the autophagy pathway, then finally advanced degradation of apoptotic cells
    • This means that initial fluorescence will be high, then decrease as time goes on
      • Slope of the line! 10-40 is positive slope, 40-80 is negative slope
  • After phagocytosis of apoptotic cells
    • Engulfed apoptotic cell becomes a phagosome and fuses with a lysosome and begin to degrade into smaller pieces
    • Phagosomes are endocytic vesicles containing the apoptotic cell
    • After fusion with a lysosome, the contents of the vesicle is degraded
  • Effectors influence on rate of efferocytosis
    • Increase activated Rac-actin reorganization (actin pushes membrane to engulf cell)
    • Enhanced signaling to find apoptotic cells
    • Increase synthesis of scavenger receptors on live cells or bridging molecules