bio lab writing

what is efferocytosis (braod)

• definition: phagocytotic removal of apoptotic cells

• who does it: Professional phagocytes (macrophages, dendritic cells)

• why it matters:

  • Apoptosis required in development

  • Turn over dead/damage cells

  • Prevents secondary necrosis + switches phagocyte to pro-resolving phenotype (limits inflammation / promotes healing)

• defective efferocytosis: apoptotic cells accumulate + undergo secondary necrosis → raises inflammation

  • Can be cause, result, or part of feedback loop that worsens disease (ex: diabetes, atherosclerosis, cancer, autoimmune disease)

  • disease → defective efferocytosis → inflammation → more disease

explain diabetes, including type 1 vs type 2

• definition: chronic disease where blood sugar stays too high

• glucose/insulin background

  • Glucose gets into cells through transporters (GLUT 1-4)

  • GLUT4 moves glucose from blood into muscle/fat cells - insulin dependent (bind insulin to make conformational change)

type 1 vs type 2

• Type 1: autoimmune attack on insulin-making cells of pancreas

  • body produces little to no insulin

  • Develops suddenly

• Type 2: gradual, linked to lifestyle

  • 1) Muscle + fat become insulin resistant

  • 2) pancreas compensates with more insulin for a while (pre-diabetes)

  • 3) pancreas eventually fails + glucose can no longer be controlled

how does efferocytosis relate to diabetes

Efferocytosis significantly impaired in uncontrolled diabetes (chronic hyperglycemia)

Consequences:

• chronic non-healing wounds (diabetic foot ulcer)

• accelerated atherosclerosis and heart disease

• retinopathy (vision loss)

• nephropathy (kidney disease)

explain macrophages (broadly)

→ versatile immune “clean-up crew” cells

• Engulf pathogens (dead cells, damaged/cancerous cells, extracellular debris) + destroy them

• Aid tissue repair

• Secrete cytokines to signal other cells

• Highly adaptable - signals they receive (mostly cytokines) tell them whether it is time to fight or time to clean up

explain M1 vs M2

M1 (inflammatory)

• Function: killing pathogens (recruit immune cells) and clearing acute debris

• persistent inflammation, phagocytosis, tissue degradation, bactericidal activity, degrading pathogens

• Efferocytosis low

M2 (anti-inflammatory)

• Function: clearing dying cells and repairing tissue/matrix

• Function: immunoregulation, fibroblast proliferation, matrix deposition, angiogenesis, tissue repair

• Efferocytosis high

what are the “find me” signals/receptors

• LPC: phosphatidylcholine cleaved by a lipase activated by caspase-3

  • Recognized by G2A receptor

• Fractalkine: found in membrane, released in membrane blebs

  • Recognized by Cx3Cr1 receptor

• ATP/UTP: released thru Panx1 channel - caspase-3 and 7 cleave the C-terminal tail of Panx1 → changing its conformation and opening channel

  • Recognized by P2Y2 receptor

explain steps for 1) recruitment/find me

1. Apoptotic cells release chemoattractants (signals) the diffuse outward + form a concentration gradient

2. Chemoattractant receptors = GPCRs are evenly spread over macrophage surface —> more ligand binds on the side facing the highest concentration

3. Binding causes conformational change in GPCR → GDP exchanged for GTP on Gα subunit

4. Active G protein dissociates into Gα and Gβγ

5. Gβγ recruits GEFs to plasma membrane

6. ELMO/DOCK complex (key GEF) catalyze GDP→GTP on Rac (small GTPase) → converting it to active state

   1. Happens mostly on leading edge → Rac stays localized, forming front

7. Active Rac1 activates Arp2/3 complex - builds new branched actin filaments → pushes out membrane as lamellipodium

CRAWLING

1. Lamellipodium protruding at leading edge anchors to surface through focal contacts (that contain integrins)

2. Rest of cell drags itself forward by traction on those anchor points

3. At trailing edge, contractile filaments (myosin II + actin) form → trailing end retracts → bulk of cell is pushed forward

what are “eat me” signals/receptors

SIGNALS

• Phosphatidylserine (PS): 

  • In living cells: PS sits on inner leaflet of plasma membrane - maintained by flippases

  • In apoptosis: flipasses are inactivated, scramlases are activated → exposing PS on outer surface

• ICAM3

RECEPTORS

• Direct: binds PS itself

  • Ex: Stabilin-2, Tim-4

• Indirect: binds PS through bridging molecule (Gas6 or ProS)

  • Tyro3: uses Gas6 OR ProS

  • Axl: uses Gas6

  • Mertk: uses ProS

explain steps for 2) recognition/eat me

1. Apoptotic cells display eat me signals

2. Eat-me receptors bind PS → signal through CrkII (adaptor protein - if Mertk)→ DOCK/ELMO → Active Rac1

explain steps for 3) internalization

1. Activated Rac1 promotes actin polymerization → deforms plasma membrane into pseudopodia that forms phagocytic cup

2. Pseudopods grow like a zipper - receptors on pseudopod bind ligands on apoptotic cell step by step

3. Pseudopodia surround target - membrane contact + fuse - apoptotic cell is internalized

explain steps for 4) digestion

1. When apoptotic cell exposes PS - Tim-4 (receptor) on macrophage binds it → drives engulfment  starts LC3-associated phagocytosis (LAP)

2. Tim-4 binding recruits PI3K complex to new phagosome: Beclin, UVRAG, VPS34, Rubicon

   1. Rubicon:

      1. Blocks canonical autophagy

      2. Activates PI3k complex

      3. Promotes production of PI(3)P (essential signaling lipid) on phagosome membrane 

3. PI(3)P stabilizes NADPH oxidase complex → generates ROS

4. ROS leads to activation of autophagy-related proteins (ATGs) + cleavage/lipidation of LC3-I into LC3-II on phagosome membrane

   1. LC3-associated phagosome = LAPosome

5. LC3-II promotes fusion of LAPosome with lysosome → forming phagolysosome

6. Low pH disrupts protein structure/apoptotic membrane + activates proteases/lipases

   1. NADPH oxidase-derived ROS oxidize and damage lipids, DNA, proteins

7. transcription factor PPARγ turns on anti-inflammatory cytokines (IL-10, TGF-β) + turns off IL-12

what are possible genes to look at

• Find-Me Receptors: P2Y2, G2A 

• Eat-Me Receptors: MerTK, Axl, Tyro3 

• Eat-Me Bridging Receptor: Gas6 

• Macrophage Movement: Rac1 

• LAPosome Formation: Rubicon

• SPM Receptor: Fpr2

  • Matters if adding effectors (lipoxin/resolvin)

• Pro-inflammatory Cytokines: TNF⍺

  • Triggered when cells sense DAMPS (damage molecules from healthy cells) or PAMPS (pathogen molecules) → produce signs of acute inflammation 

  • Push towards M1

  • High in diabetic conditions

• Anti-inflammatory Cytokines: TGFβ, IL-10, IL-4

  • Block pro-inflammatory cytokine action/release, push cells toward M2, act more directly on efferocytosis

  • Low in diabetic conditions

• Protease that cleaves MerTK: ADAM9

  • High in diabetic conditions

ALL are better if more of it EXCEPT TNF⍺/ADAM9

what are possible proteins to look at

• Find-Me Receptors: S1P-R1 (intracellular as second messenger or on the PM as receptor) 

  • Receptor for find me protein S1P

  • Low plasma membrane expression / high intracellular pool (amount): trafficking defect - leaving macrophage “blind” to find-me gradients

  • Overall downregulation: severe impairment in find me step

• Eat-Me Receptors: MerTK, Axl, Tyro3 

  • Decreased total expression: macrophages have lost their primary receptors for recognizing dead cells, maintaining a pro-inflammatory phenotype

  • Could be cleaved by ADAM9

• Macrophage Movement: Actin (Phalloidin)

  • Weak phallodin rings: mechanical engulfment failure - cytoskeleton lacks the plasticity to pull the target in

  • hyper-polymerized/rigid cytoskeleton: chronic metabolic inflammation, preventing the dynamic remodeling required for effective particle internalization

• Transcription Factor: PPAR𝛾 - suppresses inflammation

  • High in cytoplasm / low in nucleus: represents failure of activation

  • High in nucleus → expect more IL-10 and TGF-β

• SPM Receptor: FPR2

ALL are better if more of it

explain qPCR calculations

• Housekeeping gene = GAPDH

• CT: cycle at which fluorescence crosses threshold

  • Lower CT = more starting transcript

1. ΔCT (within one group) → ΔCT = CT (target gene) – CT (housekeeping gene)

• Normalizes target gene to housekeeping/reference gene - corrects for differences in starting materials

• Do it for treatment group and control for EACH target gene

2. ΔΔCT (across groups) -> ΔΔCT = ΔCT (treatment) - ΔCT (control)

• Compares normalized expression in treatment group against control group

3. RQ (relative quantification) → RQ = 2^(-ΔΔCT)

• Converts to a linear fold change that is easy to read

• RQ=1: no change in target gene expression vs control

• RQ >1: expression increased versus control

• RQ<1: expression decreased versus control