Lecture Notes on Cell Biology

Protein Sorting

• Proteins must be sorted and distributed to specific locations within the cell.

• This process relies on signal sequences.

• Signal sequence:

  • Directs proteins to their correct organelle.

  • Different proteins have different sequences for different destinations.

Import and Export Sequences

• Signal sequences facilitate entry (import) and exit (export) from organelles.

• Import sequence: A signal sequence that gets a protein into an organelle.

• Export sequence: A signal sequence that allows a protein to leave an organelle; involves revealing a sequence after another is removed.

Strategies for Protein Sorting

• Three main strategies for protein sorting:

  • Pores

  • Vesicles

  • Translocators

• Translocator: Acts like a tunnel.

• Pore: Acts like a hole.

• Vesicle: Carries cargo around the cell.

• Each strategy requires a specific signal sequence.

Pores

• Associated organelle: Nucleus.

• Pore mechanism: Nuclear Pore Complex (NPC).

• NPC:

  • A complex of proteins.

  • Made of nucleoporins (proteins that form the pore).

Nuclear Pore Complex (NPC) Structure

• Cytosolic fibrils:

  • Spine-like structures extending outwards from the NPC.

  • Function: Tethering proteins that are trying to enter the nucleus.

Nuclear Import

• Nuclear Import Receptor (NIR):

  • Surveils the outside of the nucleus.

  • Recognizes and binds to cargo proteins containing a Nuclear Localization Sequence (NLS).

• Nuclear Localization Sequence (NLS):

  • An import sequence that signals entry into the nucleus.

• Ran:

  • Helps facilitate the movement of the NIR and its cargo protein across the pore.

  • Initially bound to GDP.

Ran-Dependent Shuttling System

• Entry:

  • Ran-GDP helps NIR with protein cross the pore.

• In the Nucleus:

  • Ran GEF (Guanine Exchange Factor) switches GDP to GTP on Ran.

  • This causes the NIR and the protein of interest to separate.

  • The protein of interest stays in the nucleus.

• Exit:

  • NIR leaves the nucleus bound to Ran-GTP.

• Outside the Nucleus:

  • Ran-GAP (GTPase Activating Protein) hydrolyzes GTP to GDP on Ran.

  • Ran becomes GDP-bound again and is ready to help NIR grab another protein.

Key Players

• Ran GEF (Guanine Exchange Factor):

  • Waits inside the nucleus.

  • Switches GDP to GTP.

• Ran GAP (GTPase Activating Protein):

  • Waits outside the nucleus.

  • Converts GTP to GDP, which means that it is a GTPase-activating protein.

Translocators

• Two methods for translocators:

  • Mitochondria.

  • Endoplasmic Reticulum (ER).

Mitochondria

• Proteins must cross two membranes to enter the mitochondria, requiring two translocators.

• Mitochondrial import sequence: A specific import sequence.

• Mitochondrial import receptor: Located on the mitochondrial outer membrane and grabs the protein.

• Protein must be unfolded (linearized) before entering the translocator.

Process

1. Mitochondrial import receptor grabs the mitochondrial protein.

2. The protein goes through translocator one on the outer membrane.

3. The protein is now in between the outer and inner membranes.

4. The protein then goes across the second translocator two.

5. The protein is now in the mitochondrial matrix.

6. In the Matrix:

   • The protein gets refolded with the help of chaperones.

   • The mitochondrial import sequence is removed by signal peptidase.

   • The protein forms a three-dimensional (tertiary) structure.

Endoplasmic Reticulum (ER)

• Protein goes via a translocator.

• The newly synthesized polypeptide emerges from the ribosome.

• ER import sequence: A sequence that signals entry into the ER.

• Signal Recognition Particle (SRP):

  • Recognizes the ER import sequence on the polypeptide.

  • Brings the polypeptide to the SRP receptor.

  • The polypeptide is already linear, no unfolding is required.

Process

1. SRP recognizes the ER import sequence.

2. SRP brings the polypeptide to the SRP receptor.

3. The complex is passed to the translocator.

4. The protein enters through the translocator and goes all the way through into the lumen.

5. In the Lumen:

   • The signal sequence is cleaved off by signal peptidase.

Start-Stop Transfer Theory

• If the signal sequence is removed and another sequence is revealed, the protein stops in the ER membrane; known as the Stop Sequence.

• The initial signal sequence is referred to as the Start Sequence.

• This process is called start-stop transfer theory.

Vesicles

• Made of a phospholipid bilayer.

• Contain cargo inside (e.g., proteins, neurotransmitters, lipids).

• Used to transport proteins from one organelle to another.

• Examples: ER to Golgi, through the Golgi, and then to the plasma membrane.

• Process: Sequential budding and fusion across different compartments.

Golgi Apparatus

• Has polarity:

  • Cis face: The side facing the ER.

  • Trans face: The side where things are leaving.

• Vesicle Budding and Fusion: Repeated process across the Golgi.

KDEL Sequence

• ER retention signal.

• If a protein with a KDEL sequence ends up in the Golgi, it is put into a retrieval vesicle and sent back to the ER.

Budding

• Goal: Create a vesicle and load it with cargo.

• Cargo Receptors: Chairs that cargo sits on.

• Adapter Protein and Clathrin:

  • Clathrin: Forms a cage around the vesicle.

  • Clathrin-coated vesicle: A common type of vesicle.

Process

1. Vesicle starts to form and is loaded with cargo.

2. Dynamin cuts the vesicle, separating it from the membrane.

3. Shedding/Uncoating: Clathrin and adapter proteins come off the vesicle and are reused.

Fusion

• The vesicle goes to a target membrane.

• Two proteins on the vesicle: VAMPs (v-SNAREs) and Rab.

  • VAMPs: Vesicle-associated membrane proteins.

• Two proteins on the target membrane: T-SNAREs and tethering protein.

Process

1. Tethering: Rab grabs tethering protein.

2. Docking: Tethering protein folds back on itself, bringing v-SNARE and t-SNARE closer together.

3. Fusion: v-SNARE and t-SNARE lock together and undergo a conformational change, triggering fusion.

Cytoskeleton

• Provides structure, protection, and support to the cell.

• Enables transportation, crawling, and compartmentalization of organelles.

• Three main protein groups:

  • Intermediate Filaments

  • Actin Filaments

  • Microtubules

Intermediate Filaments

• Strong, tough, sturdy, and rope-like protein complexes.

• Made of building blocks, including single polypeptides that form monomers, dimers, and polymers to form a larger filament.

• Four classes:

  • Lamins: Found in the nucleus.

  • Keratin: Found in epithelial cells, hair, skin, and nails.

  • Neurofilaments: Found in nerves.

  • Vimentin: Cells, connective tissue, muscle.
    *Diseases: Skin, hair, neural issues (if intermediate filaments misbehave depending where they are at).
    *Nuclear: Lamins.
    *Cytosolic: Keratin, neurofilaments, vimentin.

Regulation

• Regulated by kinases and phosphatases.

• Phosphorylation and dephosphorylation regulate the buildup and breakdown of intermediate filaments.

Microtubules

• Important for intracellular transport (highways).

• Form mitotic spindles during cell division.

• Make up cilia (motile and non-motile).

• Made of tubulin building blocks.

• Can be built up and broken down.

• Dynamic Instability: The ability to build and shrink microtubules.

• Microtubule Associated Proteins (MAPs): Regulate growth and shrinking.
  Additions of Alpha + Beta = Microtubules growing and if peeled off = they are shrinking.

Dynamic Instability

• Adding or peeling off is based on alpha, beta dimers.
  *Growing: add alpha, beta, alpha, beta, alpha beta (Build).
  Shrinking: they are peeled off (Shrink).

• Building blocks: Tubulins with GTP attached.

• As the tubulins are incorporated into the growing microtubule, GTP is hydrolyzed to GDP.

• Shrinking microtubules have GDP-bound tubulins.

Microtubule Associated Proteins (MAPs)

• Regulate growth and shrinking.

• Examples:

  • MAP2

  • Tau: Stabilizing map that helps neuronal axonal microtubule axonal bridges stay there;

Centrosome

• Microtubules originate from the centrosome.

• Gamma tubulin ring complex: The base where microtubules extend from.

Actin Filaments

• Involved in muscle contraction, cell crawling, and contractile ring formation during M phase.

• F-actin (large filaments) are made of actin monomers.

• Treadmilling: Adding monomers to one side and removing them from the other.

• Ability to grow and shrink is highly dynamic.

• Adding monomers with ATP attached.

• Removing monomers with ADP attached.

Actin Binding Proteins

• Regulate the length and stability of actin filaments.

• Example: Myosin (interacts with actin for muscle contraction).

• Three involved in growth:

  • Thymosin:Limits actin filament growth.

  • Profilin:Limits actin filament growth.

  • Formin: Growth of actin filament.

• Filamin:Cross link.

Cell Crawling

• Leading edge: Structure used by cells to crawl.

• Lamellopodia and filopodia: Actin-rich regions in the leading edge that extend and retract.

  • Lamellopodia:

    • My knuckles

  • Filopodia: Fingertips regions.

• Treadmilling enables cells to crawl, stop, and change direction.

Cell Cycle

• Function: To prepare for cell division and divide into two daughter cells.

• Importance: Ensures safe and proper division to prevent diseases like cancer.

• Two phases: M phase and Interphase.

• Interphase: G1, S, G2

Goals of Each Phase

G1 Phase: The Goals/Functions is to grow, observe any DNA and the repair the DNA damage.
*Pausing, repairing (And that's specific to the mechanism).
-Spot the problem that there is a double stranded break.
-Pause so we don't duplicate all this stuff because the dna duplicate.
-Repair DNA
-If we can’t repair we go to apoptosis.
-If Everything looks it go to S. (Everything starts duplicating)

S Phase:

G2 Phase: The Goal/Functions of G2 is to growth, increasing metabolic actively, protein synthesis. What types of proteins you may start making are Tubulins (Make to maintain Mitotic Spindles: M Phase).
If you’re not sure what each is prepping for the next phase.
*The Logic you build it, build it or something that’s good gonna be squashed.
-increase metabolism.
-protein synthesis.
Tubulins: (m phase) of the chromosomes in mitotic spindles….
So that’s the cell division part.
ACTIN: because why would you produce actin make the contractile ring.
-Some mitotic and some are for cytokinesis.
Regulatory Protein/ Checkpoint:
Cyclin CDK

What phase does Cyclin Oscillate (if it goes up you transcribe and translate if it goes down its degrading(ubiquitiinating Chain))??!!!

Mechanisms

Cyclin CDK switch on and off
-How you can got red of them
G1 Positive mechanism
M Phase: Then Interphase Then M Phase Then Interphase

When you are trying to push from G2 to M you need to increase cyclin CDK that involved in mitosis.
Then activate through transcription and translation, and then it helps push into M( then come out of M (break the cyclone.)
Add ( APC Anaphase Promoting complex).This will add the (AP chain- and will go to the Proteasome and if it does what happens the kinase to it switch off).
Because the kinase depends on cylclome so imagen you’ve got kinases doing stuff that the cyclones on. It jumps off.
The thing to pull off this kinase her is this ubiquitination/ap.
Mechanism #2:
Cycle-CDK/ Then we1 comes in to ADD (An Inhibitory Phosphate) to the complex ( but your still ready( not fully activated). When C-DC/25 (Comes In) ( the phoshatase come in its like go!!).
But we does is the no.
-So there are is that three steep process to wake up these cyclones on cdk.
-Example of Something in what is involved in mitosis.
Mechanism with G1