Lysosomes, Mitochondria, and Cellular Processes Overview

Lysosomes

Organelles containing hydrolytic enzymes for digestion

single membrane organelle

uses 50+ hydrolytic enzymes

active at pH=5

De Duve

Scientist who discovered lysosomes.

how were lysosomes discovered

Centrifugation: Technique to separate organelles based on density.

organelle purification

Hydrolytic enzymes

Enzymes that catalyze the breakdown of molecules.

What distinguishes lysosomes from other organelles

lysosomes have an acid phosphatase (a marker enzyme)

what is a marker enzyme

enzyme found in one and only one organelle to serve as a means of identification

3 functions of lysosome

1. heterophagy

2. autophagy

3. endocytosis

Heterophagy

Digestion of external substances by lysosomes.

Autophagy

Self-digestion of cellular components by lysosomes.

Endocytosis

Process of engulfing substances into the cell.

Primary lysosomes (heterophagy)

Lysosomes before fusion with late endosomes.

Secondary lysosomes (heterophagy)

Lysosomes after fusion with late endosomes.

Virchow

Historian who studied white blood cells

Examined pus and saw that WBC with RBC inside

said that white blood cells were giving birth to red blood cells

Metchnikoff

Father of probiotics

said that WBC were not giving birth

WBC are actually undergoing phagocytosis

Opsonization (metchnikoff)

Coating bacteria with antibodies to enhance phagocytosis.

Abs bind to FC receptors and WBC

WBC undergoes endocytosis of bacteria

lysosomes in WBC destroy bacteria

Myasthenia Gravis

autoimmune disorder where immune system attacks muscular junction/neuromuscular transmission

Causes of myasthenia Gravis

Many causes but mainly due to ACh being targeted by Abs and can’t bind to AChR or because AChR is damaged

AChR

Acetylcholine receptor targeted in Myasthenia Gravis.

Droopy eyelids

Common symptom of Myasthenia Gravis.

Experiment for myasthenia gravis

Curare: Plant derivative that blocks acetylcholine receptors.

People with MG more sensitive to this derivative than healthy people (i) When ACh can’t bind to receptor, muscle contraction and movement is inhibited (ii) This is already the case for people with MG so curare makes it worse (b) Acetylcholinesterase (AChE)inhibitors (example: eserine) increases ACh availability, allowing for relief for patients with MG

AChE inhibitors

Drugs that increase acetylcholine availability.

Miniature end plate potential (MEPP)

Measurement of ACh in neurotransmitter vesicles.

2mV for heathy ppl

1mV for MG pts

3H alpha bungarotoxin (experiment for MG)

Radioactive substance used for ultrastructural autoradiography

• Scientists labeled α-bungarotoxin with radioactive isotopes or fluorescent dyes.

• They used it to visualize AChRs at neuromuscular junctions under microscopes.

showed less AChR in MG patients than healthy ppl

Electroplax (experiment for MG)

Part of electric stingray with high AChR concentration.

Researchers used electroplax tissue from electric fish to extract large amounts of pure acetylcholine receptors.

AChR purified from electroplax and added into mice that were injected with serum from MG patients (c) Mice showed MG symptoms showing that just adding AChR won’t reduce MG symptoms (i) Anti-AChR Abs will bind to the AChR and induce heterophagy causing lysosomes to degrade and prevent them from being used

what did all of the experiments for MG show us

These experiments showed that MG is associated with low functioning ACh and AChR levels

AChR

Acetylcholine receptor, crucial for neuromuscular transmission.

Anti-AChR Abs

Antibodies that bind AChR, impairing function.

Heterophagy***

Process where lysosomes degrade unwanted cellular components.

Neostigmine (treatment for MG)

Inhibits acetylcholinesterase (ACh esterase), increases Ach half life

In MG, the problem is that ACh can’t activate enough receptors (because antibodies have blocked, destroyed, or reduced them).
So if ACh is broken down too quickly by AChE, there’s even less chance it’ll find and activate the few functioning receptors that are left.

Prednisolone (treatment for MG)

Corticosteroid that decreases immune response

Vyvgart (treatment for MG)

Blocks IgG binding to ACh, preventing degradation***

Capillary endothelial cells bind to IgG molecules (i) This prevents them from binding to ACh (a) ACh gets degraded when IgG molecules bind to them

Autophagy

Cellular process for degrading and recycling organelles.

Yoshinori Osumi

Scientist who discovered autophagosomes (a double-membrane vesicle formed during autophagy, a cellular process where the cell breaks down and recycles damaged or unnecessary components. It engulfs cytoplasmic material and delivers it to lysosomes for degradation.

Responsible for organelle turnover a) When organelles die autophagy destroys it b) M.T half life 5-6 days c) Pathway

Phagophore

Membrane structure initiating the autophagy process.

(a) Membrane bound

(b) Starts autophagy

(c) Comes from RER

emerges from the RER and becomes autophagosome

LC3 proteins

Regulate formation of autophagosomes.

many druges will mess with autophagosomes

Chloroquine

Drug affecting autophagy

Can make cancer cells more sensitive to cancer drugs but can also harm normal cells

Myopathy (side effect of chloroquine)

Muscle disease causing decreased strength.

Retinopathy (side effect of chloroquine)

Damage to the retina, affecting vision.

Silicosis (lysomal disease, nongenetic/environmental)

Also known as Miner’s disease

Lung disease caused by silica being inhaled in the coal mines

causes fibrosis

Fibrosis

Thickening and scarring of connective tissue.

(i) Increased collagen being released in the lungs (ii) Lungs become less elastic and decrease CO2 O2 exchange

experiment done for silicosis/fibrosis

(i) Cells placed in culture with silica which is naturally sharp (ii) Cells will endocytose the silica and the lysosomes used to transport them will puncture and become leaky and the cell dies due to hydrolytic enzymes spilling out (a) Can be resolved via collagen

Inherited lysomal storage diseases

about 70 different types

most common treatment is enzyme replacement therapy (ERT)

Tay Sachs (inherited lysomal disease)

Lysosomal storage disease due to Hex A deficiency.

Ganglioside GM2 builds up in lysosomes

symptoms of Tay Sachs

(a) Hypotonia

(b) Red spots in the retina

(c) Startled by loud noises

(d) Blindness

(e) Deafness

NO ERT YET BUT GENE THERAPY HAS BEEN SUCCESSFUL

Hurler syndrome (inherited lysomal disease)

Genetic disorder caused by alpha-L iduronidase deficiency.

Those with this syndrome look like gargoyles

Aldurazyme is enzyme replacement therapy for hurler syndrome

Experiment for Hurler syndrome

(a) Hurler cells placed on top of culture (b) Normal cells on bottom (c) Over time hurler cells started to look like normal cells (d) This experiment was the basis for ERT

I cell disease (inherited lysomal disease)

Very rare

caused by failure to add M6P

• M6P is a tag that gets added to certain enzymes in the Golgi apparatus.

• This tag is like a shipping label — it tells the cell to send those enzymes to the lysosome, where they’re needed to break down various substances

Symptoms of I cell disease

same as Hurler but more severe

skeletal abnormalities

I cell disease experiment (figure out)

(a) If someone knows what the experiment was that would be great (b) What was observed (i) I cells have lysosomes without hydrolytic enzymes (ii) (iii) Excess lysosomal enzymes were found outside the cell I cells can endocytose lysosomal enzymes (a) Fibroblasts can’t

What we know from I cell disease experiment

Because of all of this we know that the N-acetyl glucosamine pathway in the cis golgi is what is affected by this disease

Gaucher disease (inherited lysomal disease)

Condition caused by glucocerebrosidase deficiency

Cerezyme is the ERT

Cerezyme

Enzyme replacement therapy for Gaucher disease.

Pompe disease (inherited lysomal disease)

Disorder from alpha-glucosidase deficiency, affecting muscles

(a) Normally converts glycogen to glucose (b) Without it, there is really only glycogen (c) Glycogen stays in lysosomes until converted to glucose (d) Muscle cells require lots of glucose (2) Causes giant lysosomes in muscle cells

Proteasomes

Degradative organelles

1. Degrades ubiquitinated protein (UPS pathway) 2. Over 30k types 3. 750,000 daltons 4. Experimentation showed that yeast cells without proteasomes died

Velcade (experiment with proteasomes

Proteasome inhibitor used to treat cancer

• Velcade inhibits the 26S proteasome, preventing protein breakdown.

• This causes a toxic buildup of proteins inside the cell.

• Cancer cells, especially myeloma cells, are very sensitive to this stress and undergo apoptosis (programmed cell death).

Localized energy control

Efficient management of energy production in cells.

Mitochondria

1. can appear as many shapes

-Co sediments with lysosomes

2. Not uniformly distributed in the cell

- Cell 1 can have MT on the right side and cell 2 can have it on the left

3. MT attached to microtubules

4. MT DNA has 13-37 genes

5. MT consists of nuclear encoded proteins

HEME (protein inside MT)

(1) HEME synthesized by MT DNA, shipped out the MT, modified outside the MT, brought back in the MT b) Why haven’t MT lost their genes? (1) Efficient localized control of energy production (2) Evolution favored these genes

Heme plays a crucial role in the mitochondria by enabling electron transport and energy production. It is a key component of cytochromes in the electron transport chain, where it helps transfer electrons and drive ATP synthesis. Heme also binds and reduces oxygen at Complex IV, allowing cells to safely use oxygen without generating harmful reactive oxygen species

Mitochondrial structure

Includes outer and inner membranes with distinct properties

Outer membrane

Contains few proteins and is highly porous.

Porin

Allows molecules under 5,000 daltons to enter the outer membrane

Inner membrane

Rich in proteins, crucial for energy processes

ETC

5x the surface area compared to outer membrane

Cardiolipin

A four-legged fatty acid

Increases electrical resistance of the membrane

Fission

a) A way to distribute MT to daughter cells during mitosis

b) A way damaged parts of MT can be removed

Fusion

a) Maintains a homogenous pool of MT in the cell

b) Could restore function of damaged MT

c) Plays a role in where MT is located in the cell

ER mitochondria associated membranes (MAM)

Regulates mitochondrial fission and lipid metabolism

a) Deals with

(1) MT fission

(2) Ca2+ homeostasis

(3) Regulates lipid metabolism, autophagy and mitophagy

(4) Alzheimers and parkinsons

Nanotubes

Facilitate mitochondrial transport between two different cells.

Mitochondrial permeability transition pore (MPTP)

Opens under stress (extrinsic), triggering apoptosis via cytochrome C.

(1) Note: this is extrinsic cell death because it is caused by an external stressor/factor

(2) MPTP is closed and cytochrome C is located inside the MT

(3) Stress causes MPTP to open and cytochrome C goes into cell cytoplasm and binds to APAF1

(4) APAF1 binds to procaspase 9 and activates it to become caspase 9

(5) Caspase 9 now activates procaspase 3

(6) Procaspase 3 gets activated and turns to caspase 3 which is known as an executor enzyme

(7) Cell undergoes apoptosis

Cytochrome C

Releases from mitochondria to activate apoptosis pathway.

Caspase 9

Activates procaspase 3, leading to cell death.

MAIN TRIGGER

Bcl2

Anti-apoptotic protein linked to cancer development.

First discovered in B cell lymphoma and is being looked at as a target drug for cancers

cyclosporin A

inhibits MPTP

targets MPTP

used to transport STEM cells

Genasense

Bcl2 antisense used to prevent Bcl2 production by targeting the mRNA

• Many cancers (like melanoma, leukemia) overexpress BCL-2 to avoid dying

• Genasense binds BCL-2 mRNA and blocks translation, reducing BCL-2 protein levels

• This should promote apoptosis in tumor cells

Obatoclax

Inhibitor of Bcl2, used in cancer therapy.

Venclexta

Treats chronic lymphocytic leukemia by targeting Bcl2.

Elesclomol

(1) Increases oxidative stress in MT by targeting ETC and causing apoptosis

2. Most types of cancer overexpress Bcl2

3. Bcl2 is an anti-apoptotic protein

Warburg effect

1. Cells use glycolysis over the MT even when there is oxygen

2. Glycolysis is optimized to get the most ATP a) More than cellular respiration

3. This is one of the reasons why cancer cells live a long time

4. Warburg effect is why HEPG2 cell line behaves differently a) Overexpression of GLUT 1 and 3 b) Overexpression of LDH c) Less processes in MT matrix

Mitochondrial diseases

1. Around 250 of them

2. Inherited solely through mother

3. mtDNA does not use correction enzymes

4. Large link between mtDNA mutations and autism

5. MT dysfunction linked with alzheimers

- a) Defect in TCA allows for beta amyloid build up which causes alzheimers

Glycolysis

Produces 2 ATP and NADH from glucose breakdown

NADH from cytoplasm gets converted to ATP via 2 antiporters

ETC inhibitors (drug that targets the MT)

Substances like cyanide that block electron transport.

CO

Proton uncouplers

Compromise H+ gradient, affecting ATP synthesis.

Proton uncouplers are molecules that disrupt the link between the electron transport chain (ETC) and ATP production in mitochondria.

DNP (proton uncoupler)

(1) Lipid soluble

(2) Weak acid

(3) Shuttles H+

(4) Proton ionophore

(5) Used to be used for weight loss

(a) OG ozempic

(b) People burned up and died when taking it

UCP-1 (proton uncoupler)

(a) Natural analog of DNP

(b) Compromises H+ gradient

(c) Thermogenin

(i) Drug that uses UCP-1

(ii) Helps warm up shivering babies

valinomycin/gramicidin (protein uncoupler)

(1) K+ ionophore

(a) Compromises MT transmembrane voltage of 200mV

Oligomycin (protein uncoupler)

Blocks flow of H+ in oxidative phosphorylation

PS inhibitors (protein uncoupler)

(1) Cycloheximide

(a) Blocks nuclear translation

(2) Tetracycline

(a) Blocks MT translation

Cellular respiration

Process converting glucose into ATP via multiple stages.

cellular respiration process

a) Glycolysis

(1) Happens in cytoplasm

(2) Glucose broken down into 2 pyruvate using ATP

(3) Net gain of 2 ATP and 2 NADH

pyruvate oxidation

(1) Pyruvate goes into MT via antiporters

(2) Will be decarboxylated to form CO2 as byproduct

(3) Will be dehydrogenated by NAD+ to form NADH

(4) CoA will bind to form Acetyl-CoA

Pyruvate oxidation turns pyruvate into Acetyl-CoA, releases CO₂, and makes NADH — it's the link between glycolysis and the Krebs cycle.

Krebs cycle

Converts Acetyl-CoA into 2 CO2, 3 NADH, 1 FADH, and 1 GTP or ATP.

The Krebs cycle (also called the citric acid cycle or TCA cycle) is the part of cellular respiration that produces energy by breaking down Acetyl-CoA (from food like glucose or fatty acids) and releasing carbon dioxide.

happens in mitochondira

Inside out mitochondria particles

1. We can’t use MT for experiments so we turn them inside out for experimentation

2. How to make them a) Isolate MT

b) Place in low osmolarity solution

c) MT will flip inside out

(1) MT will have F0 and F1 proteins at this point on the inside out membrane

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use pH sensitive dyes

ETC

Electron Transport Chain; crucial for ATP production.

(1) FADH and NADH release electrons through protein chain

(2) Causes shift in proton gradient

(3) H+ will go through transporters that will phosphorylate ADP into ATP

(a) ATP synthase specifically

ADP phosphorylation

Conversion of ADP to ATP via phosphate addition.

Radioactive PO4

Phosphate used to trace phosphorylation processes.

MT functionality

Mitochondrial function assessed by ATP production efficiency.

Validamycin

Inhibits voltage and ATP production in cells.

DNP

Disrupts voltage and H+ gradient, reducing ATP.

Vm

Membrane potential, additive with H+ gradient.

Mechanical agitation

Separates F0 and F1 ATP synthase components.

F0 particles

Have working ETC but no ATP synthesis.