L5 Cell Biology

🧠 Concepts Introduced

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Learning Objectives Explain the function of cellular organelles

• cytoskeleton
• mitochondria, chloroplasts, peroxisomes
• endoplasmic reticulum & Golgi apparatus Describe protein trafficking & sorting Describe the importance of endocytosis and exocytosis Understand degradation of cellular components

Cellular Organelles

cytoskeleton mitochondria, chloroplasts peroxisomes endoplasmic reticulum Golgi apparatus

percentage of nucleus is small cuz differentiated ady. stem cells nucleus can go up to 30% cuz actively differentiating

Cytoskeleton

An intricate network of protein filaments that extends throughout the cytoplasm

Functions of cytoskeleton:

• Maintain cell shape
• Maintain internal organization
• Provides mechanical support that enables cells to carry out essential functions like cell division and movement.

Three types of protein filaments

• intermediate filaments
• microtubules
• actin filaments

Each from different protein subunits:

• Fibrous proteins form the intermediate filaments
• Tubulin subunits form microtubules
• Actin subunits form actin filaments

Intermediate Filament

Strong rope-like polymers around 8-12 nm in diameter Provide mechanical stability to cells and tissues

It is grouped into four classes in cytoplasm and nucleus

Cytoplasmic

1. keratin filaments in epithelial cells in skin or hair

2. vimentin and vimentin-related filaments in connective-tissue cells, muscle cells, and glial cells

3. neurofilaments in nerve cells

Nuclear

1. nuclear lamins in all animal cells

Microtubule

Long and relatively stiff hollow tubes of protein

Can rapidly disassemble in one location and reassemble in another

Many microtubules grow out from the centrosome to form mitotic spindle and cytoskeleton throughout the cell. (spindle fibre)

Microtubules also grow from the basal body to form cilia and flagella

Microtubules are built from tubulin subunits (dimer composed of two very similar proteins called alpha tubulin and beta-tubulin, bound tightly together)

disassemble by disassembling alpha and beta tubulin

Actin Filament (or Microfilament)

Polymers arranged in a helical-chain, formed by attached actin monomers and have around 8 nm of diameter.

Form a cortical network that lines the inner surface of the plasma membrane of animal cells, granting its strength and shape.

Interact with actin-binding proteins, and control the behavior of the intact filaments

Actin filaments can also form stiff and stable structures such as microvilli on the epithelial cells lining the intestine

lamellapodium expands and actin filament supports the shape

Mitochondria

Consists of inner & outer membranes, cristae, matrix, mtDNA (16.6 kb) and ribosome

Inner membrane forms folds called cristae that converts energy provided by breakdown of nutrients in the matrix into ATP

Human mitochondrial genome encodes 13 proteins involved in oxidative phosphorylation (OXPHOS) (electron transport chain + chemiosmosis), 16S and 12S rRNAs, and 22 tRNAs, which are required for translation of the proteins encoded by the organelle genome.

80 ETC proteins are nuclear-encoded, translated in cytoplasm and transported into mitochondria.

fission and fusion Mitochondria are highly dynamic organelles that maintain their morphology via continuous fission and fusion, also known as mitochondrial dynamics.

Mitochondrial dynamics play critical roles in maintaining functional mitochondria when cells experience metabolic or environmental stresses.

Mitochondrial fission and fusion can maintain their abundance, morphology, and quality and cell function.

Mitophagy is a cellular process that selectively removes the aged and damaged mitochondria via the specific sequestration and engulfment of mitochondria for subsequent lysosomal degradation

aged mitochondria

L4 Cell Biology#ETC

1. The biological machinery of ETC isn't 100% efficient. As electrons pass between the protein complexes (specifically Complex I and Complex III), a small percentage (0.2% to 2%) accidentally "leak" out early.
2. The Creation of ROS: When these rogue, leaked electrons collide prematurely with surrounding oxygen molecules, they prematurely reduce the oxygen. This forms superoxide O2^2, which is the baseline free radical that further transforms into other highly volatile reactive oxygen species, like hydrogen peroxide H2O2 and hydroxyl radicals OH-.

In aged humans and animals, higher levels of mutations and deletions in mitochondrial DNA (mtDNA) lead to reduce mitochondrial protein expression and impairments in oxidative phosphorylation (OXPHOS), which worsens the generation of reactive oxygen species (ROS).

Mitochondria also appear fragmented and swollen while also displaying impaired mitophagy (the cellular process that clears out damaged mitochondria) and distribution of mitochondria to neuronal compartments such dendrites and axons

Mitochondrial dysfunctions include strokes, neuropathy, myopathy, hearing loss, and type 2 diabetes mellitus

! complex 1 2 3 4 for ETC is in cristae when it is folded the complexes are closer to each other compared to linear. this helps to transport electrons at a shorter distance

evolution of mitochondria one bacteria engulf another bacteria

mitochondria fission is to separate the damaged part and the healthy part will fuse with another healthy part to form a bigger healthy mitochondria

Chloroplast

stroma equivalent to matrix in mitochondria

Photosynthetic bacteria and chloroplasts of algae and plants use light-capturing green pigments, chlorophyll to produce energy

Photosynthesis - the series of light-driven reactions that creates organic molecules from atmospheric CO2

Photosynthesis occurs only during daylight hours, producing ATP and NADPH these energy carriers can convert CO2 into sugar (carbon fixation)

The inner membrane surrounds a large space called the stroma (analogous to the mitochondrial matrix)

In chloroplasts, the photosynthetic machinery (eg. light capturing systems, ETC and ATP synthase) is contained in the thylakoid membranes, NOT inner membrane

Photosynthesis

light energy + CO2 + H20 - sugars + 02 + heat energy Thylakois

Stage 1- energy from sunlight is used to produce ATP and NADPH Stage 2 - these energy carriers (ATP, NADPH) are consumed to synthesize sugars

Photosynthetic mechanisms capture energy from photons to drive two types of reactions - light & dark reactions

Light (stage 1): • Energy-producing reactions are called light reactions Energy comes from sunlight by chlorophyl Depends on continuous absorption of photons which occurs in ETC in the thylakoid membranes for generation of ATP and oxygen (similar to ETC on mitochondrial inner membrane)

Dark (stage 2): Convert carbon dioxide into 3-carbon sugar phosphate (carbon fixation) (glyceraldehyde 3-phosphate (G3P)) L4 Cell Biology#Preparatory phase (Glycolysis) This process can occur in the absence of sunlight (hence, dark reactions) However, this reaction depends on ATP and NADPH produced by light reactions, so it eventually stop in the dark Begin in chloroplast stroma, and simple sugar is exported to the cytosol

Peroxisomes

Present in all eukaryotic cells About 1 um in diameter

Organelles bounded by single lipid bilayer (mitochondria and chloroplast have double membranes) with embedded proteins and contain a dense matrix

Peroxisomes form in two ways: (1) budding of precursor vesicles from the endoplasmic reticulum or (2) growth and division of preexisting peroxisomes

(they are important for fatty acid metabolism and detoxification single lipid bilayer with no intermembrane space urate oxidase - to break down uric acid peroxisome after division (in theory) can mature and grow)

Functions of peroxisome:

1. Alpha- & Beta-oxidation (Lipid breakdown)

• Breakdown branched chain to very-long-chain chain fatty acid (alpha oxidation)
• Then by oxidation of very-long-chain-fatty acids (24-26 carbon units) into Acetyl-CoA and Acyl-CoA in liver and kidney (will change oxygen to hydrogen peroxide in the process of converting to acetyl Coa and Acyl-Coa which is toxic) (beta oxidation)

2. Biosynthesis of fatty acids

• Acetyl-CoA is used to produced cholesterol which is then transformed into bile saltand steroid hormones
• Acyl-CoA is made into phospholipids (plasmalogen)

3. Contain catalase that breakdowns hydrogen peroxide (H2O2) into two H2O and one O2

4. Ethanol metabolism involves the conversion of ethanol to acetaldehyde and then acetyl-CoA.

(function: branched chain cannot be used as source of energy as it is a long polymer so peroxisome oxidises it the oxidation removes 2 carbon each time (beta oxidation) linear polymer can branch out (branched chain) removing the branch making it linear (alpha oxidation) can be used to make special phospholipid, present in myelin in nerve cell)

Endoplasmic Reticulum (ER)

Largest membrane-delineated intracellular compartment within eukaryotic cells

The ER around cell nucleus form a double membrane bilayer barrier called nuclear envelope

ER processes most proteins and lipids that make up the membrane of cellular organelles

ER specialized for protein folding, modification and degradation is rough ER

mRNA out of nucleus to ER that's why ER is near the nucleus

to have mature protein / activated protein it has to be modified in ER that's why we say protein is syntheised in ER.

Rough ER arranges proteins into tertiary structure and also modified proteins eg. N-, O- and C-linked glycosylations

The carbohydrate can attach to:

• Nitrogen (N) of asparagine → N-linked glycosylation
• Oxygen (O) of serine or threonine → O-linked glycosylation
• Carbon (C) of tryptophan → C-linked glycosylation

Mature proteins are transported in vesicles to Golgi apparatus

Misfolded proteins are degraded in ER rather than being exported from the ER

Smooth ER composed of tubular elements lacking ribosomes, responsible for lipid biosynthesis

Acetyl CoA enters in smooth ER can be converted into fatty acids, phospholipids and cholesterol

Cells in testes and ovaries contain huge amount of smooth ER for producing testosterone, estrogen progesterone.

Golgi Apparatus

Golgi apparatus can be derived from plasma membrane, nuclear membrane or ER

It contains 4 structural components: cisternae, tubule, vesicles and Golgi vacuoles.

Stacks of Golgi cisternae exhibit cis-to-trans polarity

Function of packaging materials from ER to targeted organelle or extracellular space

Proteins and lipids from the ER enter the cis face (entry face)

Cargo leaves from the trans face at the opposite side of the stack

Then travels to Trans Golgi network (TGN) for sorting and packaging before leaving for the target sites.

Protein Trafficking

1. mRNA is transported out of the nucleus and shipped to rough ER
2. ER svnthesize the proteins through ribosomes (can also occur freely in the cytoplasm) and ship to Golgi apparatus
3. Golgi apparatus packages and label proteins by Golgi enzymes for transport (proteins are big and cannot easily move through the cell membrane) or store within the cells
4. Golgi apparatus modifies the proteins (eg. glycosylation) so that transport proteins can recognize them OR enables them to be packed into a secretory vesicle and go through exocytosis upon membrane fusion

Protein Sorting

1. For some organelles (eg. mitochondria, peroxisomes, etc), proteins are delivered directly from the cytosol; or

2. Nascent proteins may contain signal sequence or sorting signal that directs the protein to the organelles

3. Proteins with signal sequence are delivered to ER, and some are retained in ER, but most are transported to Golgi apparatus followed by plasma membrane or other organelles

4. Peroxisomes acquire some membrane proteins from ER, but most of the enzymes enter directly from the cytosol

Proteins are transported via THREE mechanisms:

1. Nuclear pores (let the entire big 3D protein in )
2. Across membranes through protein translocators (unfolding 3D proteins to linear single strands)
3. Transport vesicles (exo- & endocytosis)

ok so from my understanding there are two things one is how the protein gets ready the other is how it is transported

so it gets ready either already in the cytosol or it has to go through ER and golgi apparatus

it can be transported by nuclear pores, vessicles like endo and exo, or translocators on organelles like mitochondria

however all of them need signal sequence

Typical Signal Sequences

Signal sequences (signal peptides) are the N- terminal sorting signal that targets the linked protein to the secretory pathway in eukaryotes and prokaryotes.

About 10%-20% of eukaryotic proteome and 10% of bacterial proteome have been estimated to have the signal sequence

Protein Sorting to Rough ER

that's why can see methionine cuz it is already chopped off

1. Signal peptides of target proteins are specifically recognized by SRP (Signal Recognition Particle) as they emerge from the ribosome.

2. SRP binds to the signal sequence

3. SRP brings the ribosome to the ER by binding to a receptor on the ER surface. The receptor is associated with other proteins that make a pore.

4. The ribosome resumes translating, feeding the polypeptide through the pore and into the ER lumen. Signal peptidase cleaves off the signal sequence.

5. The completed polypeptide is released into the ER lumen

Co-translational translocation begins when the Signal Recognition Particle (SRP) binds to the hydrophobic signal sequence at the front of a growing protein chain, halting translation and docking the ribosome onto an ER membrane receptor. Once docked, the SRP detaches and recycles, allowing the ribosome to resume translation and push the unfolded protein directly into the translocon channel. Because the greasy signal sequence gets stuck like a wedge in the channel wall, the continuing chain is forced to bend and enter the ER lumen as a hairpin loop. Signal Peptidase (SPase), a permanent enzyme resident sitting at the luminal exit of the channel, then snips the junction right behind the trapped signal sequence. This cleavage frees the main body of the protein to drop completely into the ER lumen where it folds, while the discarded signal peptide remains stuck in the membrane to be broken down.

Vesicular Transport

why need a vesicle. cuz it takes a long time for protein to diffuse and can have bulk packaging of protein (transport a lot of protein at once)

Transport from ER to Golgi apparatus and to other compartments is carried out by continual budding and fusion of transport vesicles

This vesicular transport extends outward from ER to plasma membrane (exocytic), and inward from the plasma membrane to lysosomes (endocytic)

Many proteins that are transported outward undergo chemical modification such as glycosylation, phosphorylation, sulfation, and proteolytic cleavage.

1. Ribosomes assemble proteins from polypeptides entering rough ER.

2. Proteins move through the rough ER, where they are further modified.

3. Transport vesicles containing the proteins are pinched off from the rough ER.

4. Transport vesicles fuse with the membrane of the Golgi complex and the proteins are released to the inside.

5. Within the Golgi complex the proteins are further processe and stored.

6. Vesicles containing the finish proteins are pinched off from the Golgi complex.

7. Vesicles travel to the cell n er fuse with the cell membrar e, the proteins to the outside

Exocytosis & Endocytosis

Endocytosis and exocytosis is a bulk transport, materials can be taken up or ejected in bulk, without passing through the cell's plasma membrane.

Exocytosis is the reverse of endocytosis. Its purpose is to expel materials from the cell into the extracellular fluid, replenish the plasma membrane, and allow the cell to respond to signals from outside the cell.

can transport faster and a lot in one go

replenish cuz when they go out they will decrease the plasma membrane then

Exocytic pathway

expels materials from the cell to the membrane surface or into the extracellular fluid

Constitutive secretion is a mechanism cells use to maintain the plasma membrane. Newly formed proteins (eg. ECM proteins, ion channels, intergrins) are embedded in vesicles created with newly synthesized phospholipids. The vesicles are delivered to the membrane and fuse with it.

Regulated exocytosis also allows cells to respond to their environment by altering which membrane proteins are present on the cell surface.

Lysosomal secretion. The cell rids itself of waste with this mechanism. Waste material is enveloped in a membrane and fuses with the plasma membrane's interior. This fusion opens the membranous envelope on the cell's exterior, and the waste material expels into the extracellular space

Endocytic Pathway

In endocytosis, material is engulfed within an infolding of the plasma membrane and then brought into the cell within a cytoplasmic vesicle.

Phagocytosis by macrophages and neutrophils

Clathrin-mediated endocytosis (see chapter 3) to absorb membrane receptors and their ligands for recycling.

Ingested materials can be delivered to endosomes, recycled to the plasma membrane (eg. neurotransmitters) or sent to lysosomes (eg. EGF-EGFR complex) for digestion

L2 Human Physiology#Endocytosis & Exocytosis (vesicles)

Three types of endocytosis: pinocytosis, phagocytosis and receptor-mediated endocytosis.

Pinocytosis is a form of endocytosis ingesting fluids containing many solutes.

Phagocytic cell extend sheet-like projections of the plasma membrane, pseudopods that engulf the bacterium and fuse at their tips to form a phagosome

In receptor-mediated endocytosis, the particle first binds to a membrane protein receptor on the surface of the cell. Binding of the target particle induces the cell to engulf it.

Degradation of Cellular Components

Constitutive turnover is a "housekeeping" function that ensures regular replacement of older molecules with newly synthesized ones or that removes misfolded, mislocalized, or otherwise damaged molecules so that they do not hinder the function of native molecules.

Two intracellular proteolytic activities

• Lysosomes
• Proteasomes

The small protein ubiquitin targets most (though not all) molecules for degradation by proteasomes and can also target proteins for degradation by lysosomes.

Ubiquitin or a polyubiquitin chain is added posttranslationally to lysine residues on protein substrates and is recognized by the cellular machinery that targets them for proteolysis.

Lipids also undergo degradation by entering lysosomes via endocytosis or autophagy

Ubiquitylation

Ubiquitin tags molecules to let the cell know that it need to be degraded

• Also known as ubiquitination
• A post-translational modification process
• Ubiquitin is a very abundant and highly conserved protein that marks proteins for degradation by lysosomes and proteasomes
• Ubiquitin also plays other roles in DNA repair, chromosome structure and assembly of ribosomes and other complexes

Degradation by Lysosomess

any particle

Lysosomes are membrane-bound compartments that sequester ~60 hydrolases including proteases and provide a low pH environment (pH 4-5), cytoplasmic pH is 6-7

Contain at least 60 distinct hydrolytic enzymes

Lysosomes degrade more diverse substances than proteasomes

Some lysosomal enzymes degrade macromolecules and reuse the break-down monomers for new macromolecule synthesis

Lysosomes degrade substrates from outside and inside the cell

Cytoplasmic substances are delivered to lysosomes by autophagosomes through a process known as autophagy

Degradation by Proteosomes

Proteasome is a cylindrical complex, composed of a 20S core particle, and two 19S regulatory particles.

The 20S complex carries out the proteolytic activities.

The 19S particle coordinates the degradation process by recognizing the polyubiquitinated substrates, unfolding, deubiquitinating and translocating them into the interior of the 20S complex, where they are degraded to oligopeptide

The ubiquitin-dependent 26S proteasomal degradation process is the major system that mediates the degradation of damaged and short-lived regulatory proteins

degrade only protein that is in the cell not extracellular and destroy protiens that are not essential (not folded properly) and one protein at a time

19s is the one that recognised ubiquitin

alpha subunits act as barrier / gate to control what comes in it will be inserted in