☠️🌐 Apoptosis + Endomembrane System

Mitochondria & Programmed Cell Death

• Programmed cell death (apoptosis) is a normal process where cells die in a coordinated sequence

• Part of organism growth/development

• Example: Interdigital cell death causes soft tissue regression between embryonic digits in many vertebrates

Bone Morphogenetic Protein (BMP) and Apoptosis

• Bone morphogenetic protein (BMP) is a secreted protein that binds to Bmp receptors (BmpRs)

• Expression of non-active BmpRs in chicken embryonic hind limbs:

  • Greatly reduced interdigital apoptosis

  • Results in webbed feet

• Ducks don’t have mutation → Have webbed feet

Apoptosis in Plant Growth

• Apoptosis plays a role in plant growth

• Madagascar Lace Plant:

  • A type of submerged aquatic plant

  • Has mature leaves that are fenestrated (contain holes)

• Plant uses programmed cell death to generate holes in its leaves

Normal vs. Apoptotic Cells

Apoptosis is characterized by:

• Shrinkage of cell

• Blebbing (bulge/protrusion) of plasma membrane

• Fragmentation of DNA and nucleus

• Loss of attachment to other cells

• Engulfment by phagocytosis

Steps:

1. Mild convolution, chromatin compaction, and margination

   • DNA begins to unwind out of chromatin and clumps against the nuclear envelope

   • Margination: Organelles are pushed to one side of cell

2. Condensation of cytoplasm

   • Cytoplasm shrinks and becomes more compact

3. Breakup of nuclear envelope

   • Nuclear envelope begins to break down

4. Nuclear fragmentation

   • Nuclear material fragments into smaller pieces

5. Blebbing

   • Plasma membrane bulges or protrudes

6. Cell fragmentation

   • Cell breaks apart into smaller apoptotic bodies

7. Phagocytosis

   • Apoptotic bodies are engulfed by phagocytic cells

The Intrinsic (Initiated Internally) Pathway of Apoptosis

• Initiated by intracellular stimuli (e.g., genetic damage, hypoxia, virus)

• Killer proteins (e.g., Bax) cause changes in mitochondrial membrane potential (creates pores)

• Bax proteins cause changes in mitochondrial membrane potential, leading to leakage of Cytochrome c

• Bax assembles on outer mitochondrial membrane (OMM) to form a pore

• Cytochrome c is released into cytosol

• Apoptosome is formed by Cytochrome c and other proteins

• Apoptosome activates executioner caspases, leading to apoptosis

Release of Cytochrome c and Nuclear Fragmentation

• Disrupts cell adhesion

• Destroys lamins (nuclear filaments)

• Breaks down cytoskeleton

• Activates DNase (genome breakdown)

Before vs After:

• Before: Intact nucleus and cell structure

• After: Fragmentation and breakdown of cytoskeleton and nucleus during apoptosis

Caspases: Activated to carry out apoptosis

Apoptosis and Diseases

• Various diseases are directly associated with apoptosis

• In some cases, insufficient apoptosis leads to diseases like cancer (cells evade apoptosis, leading to uncontrolled cell growth)

• In other cases, excessive apoptosis causes diseases like neurodegenerative disorders (e.g., Alzheimer's, Parkinson's) where too many cells die, leading to tissue damage

Main Functions of Intracellular Compartments

• Cytosol: Protein synthesis, many metabolic pathways

• Nucleus: Contains genome, DNA, RNA synthesis, ribosome assembly

• Endoplasmic Reticulum (ER): Synthesis of lipids, synthesis of proteins

• Golgi Apparatus: Protein modification, packaging of proteins and lipids

• Lysosomes: Degradation of cellular material

• Endosomes: Sorting, recycling

• Mitochondria: ATP synthesis, apoptosis

• Chloroplasts: Photosynthesis, ATP synthesis

• Peroxisomes: Oxidation of toxic molecules

Early Electron Microscopy Observations of Cytoplasm

Membrane-Bound Organelles Identified by Early Electron Microscopy

• Endoplasmic Reticulum (ER)

• Endosomal Transport Vesicles

• Golgi Complex

• Lysosomes

• Vacuoles

Early EM of cytoplasm revealed:

• Membrane-bound organelles and vesicles

• Extensive network of membranous canals

• Stacks of cisternae (sac-like structures)

Polarized Structure of Secretory Cell

• Secreted proteins (e.g. mucin, glycoprotein in mucus) are:

  • Synthesized in rough ER

  • Processed in ER

  • Further processed in Golgi body

  • Concentrated in vesicles

  • Delivered to plasma membrane for secretion and exocytosis

• Goblet cells:

  • Produce mucigen granules (precursors of mucus)

Overview of Biosynthetic / Secretory Endomembrane System

• Nuclear envelope

• ER

• Golgi apparatus

• Vesicles

• Lysosomes

• Plasma membrane (phospholipid bilayer + proteins, cholesterol, glycolipids, glycoproteins)

• Facilitates:

  • Exo and endocytosis

Protein Synthesis - ONLY KNOW RED

Rough ER & Translation

• Ribosomes attached to rough ER synthesize proteins

• mRNA from nucleus is translated by ribosomes

• rRNA in ribosomes facilitates peptide bond formation

• Proteins enter ER lumen during translation

• In ER, proteins undergo folding and initial modifications

Transport Through Endomembrane System

• Properly folded proteins are packaged into transport vesicles

• Vesicles move from ER to Golgi apparatus

• In Golgi, proteins are further modified, sorted, and packaged

• Sorted proteins transported via vesicles to:

  • Plasma membrane (for secretion)

  • Lysosomes (for degradation enzymes)

  • Other organelles (for functional use)

Summary of Flow

1. Nucleus → mRNA

2. Ribosome on rough ER → translation

3. ER lumen → folding & modification

4. Vesicles → transport to Golgi

5. Golgi → processing & sorting

6. Vesicles → final destination (e.g. membrane, lysosome, secretion)

Technique: Using GFP to Track Cell Components

• Green Fluorescent Protein (GFP) from Aequorea victoria (jellyfish)

• GFP gene is fused with gene that codes for target protein

• Fusion protein is expressed in cells

• Allows visualization of protein's location, movement, and dynamics using fluorescence microscopy

• Common tool in cell biology for studying protein localization, organelle tracking, and cell behavior

Using GFP to Track Cell Components

• GFP fusion protein fluoresces, enabling visualization under a microscope

• Observation of fusion protein reveals information about endogenous protein

  • Localization of protein in a cell or organism

• Variants of GFP emit fluorescence at different wavelengths

  • Achieved through genetic modifications

Transport of Material Between Compartments

• Organelle → PM (and vice versa)

• Organelle → Organelle

• Uses transport vesicles (~50-75 nm)

  • Small, spherical, membrane-enclosed organelles

  • Bud off donor compartment and fuse with acceptor compartment

• Targeted movement (directed)

  • Uses cytoskeleton and motor proteins

  • Sorting signals recognized by receptors

Key Elements of Vesicle Trafficking to a Compartment

• Approach

  • Uses cytoskeleton and motor proteins

  • Can be anterograde (forward) or retrograde (backward)

• Tethering

  • Uses Rab family proteins and other specialized proteins

• Docking (SNARE assembly)

  • Vesicle has v-SNARE

  • Target membrane has t-SNARE

  • SNARE proteins interwind to form SNARE complex

  • SNARE complex pulls vesicle and target membrane closer (initiates lipid mixing)

• Fusion of vesicle and target membrane

  • Fusion allows for cargo to release

  • SNARE complex is dissembled

Protein Transport Through Endomembrane System

• 0 min: Protein starts in ER (where it’s synthesized)

• 40 min: Proteins are concentrated in Golgi apparatus for further processing

• 180 min: Proteins are transported to PM (to carry out functions)

Orientation of Transmembrane Proteins

• Orientation of transmembrane protein is maintained through its travel

• Cytoplasmic end of protein sticks out into cytosol

• ER lumen end of protein faces into lumen of ER/Golgi/vesicle

• *After exocytosis, ER lumen end of protein is on outside of plasma membrane since it merges and inverts

• Proteins are tagged with signals to allow them to go to their designated locations

Exocytosis Examples

• Organelle → Plasma membrane

• Secretion of neurotransmitter

  • Regulated exocytosis (by Ca²⁺)

  • Process:

    • Action potential reaches the axon terminal, triggering calcium ion influx

    • Calcium ions facilitate the fusion of synaptic vesicles with the presynaptic membrane

    • Neurotransmitters are released into the synaptic cleft and bind to receptors on the postsynaptic membrane

    • This binding initiates a signal in the postsynaptic cell

Endocytosis Examples - Highlighted is most important

• Plasma membrane → Organelle

• Reduces number of AMPA receptors on plasma membrane

  → Less Na+ enters neuron when signal arrives

  → Weaker synaptic response

• Arc protein
  → Controls how many AMPA receptors are in membrane

• Activity-dependent internalization of AMPA receptors

  • GluA1/2: Subunits of AMPA receptors involved in synaptic transmission

  • Endophilin: Protein helps in vesicle formation during endocytosis

  • Arc: Protein involved in synaptic plasticity, translated locally at synapse

  • Dynamin: Helps vesicle fission during endocytosis

  • Reduced AMPA-mediated transmission: AMPA receptor internalization reduces synaptic transmission

  • Synaptic activity: Activates pathways that promote AMPA receptor internalization

  • Ribosome: Local translation of Arc near synapse during activity