PHGY 170 Module 5

What is the cytoskeleton?

- A network of structural proteins found in cells

What does the cytoskeleton do?

- Permits cellular functions such as signalling and vesicular transport

- Gives the cell unique properties such as cell motility

- Defines the shape of a cell and the distribution of cellular contents

What are the three classes of structural proteins?

- Intermediate filaments

- Microtubules

- Actin

What are the primary purposes of each structural protein?

- Intermediate filaments: Add mechanical strength to cells

- Microtubules: Support trafficking within cells

- Actin: Support cellular motility or large-scale movements like contraction

What are the key features of intermediate filaments?

- Analogous to the bones of the body

- Strongest cytoskeletal filaments

- Tissue and cell specific expression

What are the major classes of intermediate filaments?

Describe the structure of intermediate filaments

- Primary: Amino acid polymer linked by peptide bonds

- Secondary: Filaments are rich in alpha helix protein structures giving them their coiled shape, hydrogen bonding prevents the stretching and collapse of filaments

- Tertiary: Coiled monomer

- Quaternary: Two coiled monomers that come together to make a coiled coil dimer. Coil structure allows for maximum hydrogen bonding. Two antiparallel (N-terminus to C-terminus) dimers assemble into a tetramer

What are the three levels of intermediate filament assembly?

- Stage 1: Unit length filament, formed by 8 tetramers, 20 nm

- Stage 2: Immature filament, formed by unit length filaments loosely interacting end-to-end, 20 nm

- Stage 3: Mature filament, compacted immature filament, 10 nm

What are the post-translational modifications of intermediate filaments? Where do they occur?

- Phosphorylation and glycosylation

- Occur in the head and tail domains of the intermediate filament subunit proteins

What is the purpose of intermediate filament phosphorylation?

- Dissolution of intermediate filaments into unit-length filaments

- Removal of phosphates by phosphatases caused filaments to reform

Why is intermediate filament disassembly important?

- For cell processes like cell division where the cytoskeleton disappears and reforms when cell division is over

What are lamins?

- Intermediate filaments found in the nucleus the forms the nuclear matrix, a dense network that protects chromatin

What are desmins?

- Intermediate filaments that connect different cellular structures together

- Important for muscle structural integrity

What is keratin?

- Intermediate filaments that bind to desmosomes to form a complex

- Makes up hair, skin, and nails

What are key features of microtubules?

- The microtubule network defines trafficking throughout the cytoplasm

- Create specific cargo routes

- Intracellular movement is not random

What are MTOCs?

- Microtubule-organizing centres

- Where microtubule assembly occurs

- E.g. centrosomes

What are α and β tubulins?

- Globular proteins that can bind GTP and bind together head to tail to form dimers

- The β-tubulin can cleave GTP to GDP and change shape when GDP is bound

Describe microtubule polymerization

- Dimers spontaneously assemble into polymers that can quickly fall apart

- Once at least 6 dimer subunits form a stable polymer it can grow laterally and longitudinally to make a protofilament

- 13 protofilaments form a sheet to assemble into a tube to act as a nucleation site for microtubule elongation

- Dimers on the ends of microtubules come and go. Microtubules shorten if disassembly occurs faster than assembly

What is microtubule assembly?

- Addition of dimers to lengthen microtubules

- Occurs when β-tubulins have GTP bound

What is microtubule disassembly?

- Depolymerization of dimers to shorten microtubules

- Occurs when the GTP on the β-tubulin is hydrolyzed into GDP causing the dimer to change conformationally and promote disassembly

How does polarity affect polymerization?

- Dimers favour assembly/binding on the plus end of microtubules

What is dynamic instability?

- The ability for microtubules to grow or shrink rapidly

Describe the features of microtubules that allow dynamic instability

- GTP cap

- GTP hydrolysis exposes GDP-bound subunits

- Catastrophic depolymerization occurs

- GTP subunits bind at one to recap the microtubule and stop depolymerization

- Microtubule begins growing again

Why is dynamic instability essential?

- Cells can rapidly explore their cytosol and create new pathways for trafficking depending on the cell's needs

- Cells can exert force. Molecules attached to microtubule ends can be rapidly transported across the cell

What is catastrophe?

- The rapid depolymerization of tubulin dimers at the plus end resulting in a shortening of the microtubule

What are the measures against microtubule catastrophe?

- Aversion: Capping

- Reversal: Rescue

Describe microtubule capping

- Plus end of microtubules are capped by capping proteins when desired length is reached.

- Adds tremendous stability

- Microtubules remain polymerized even if their dimers are in the GDP-bound form

Describe microtubule rescue

- The reversal of catastrophe is if there are enough GTP-bound dimers or other proteins present

What are microtubule-based motor proteins?

- Proteins that control trafficking by binding to cargo and walking along microtubules to transport it

- Consume ATP

Which way does kinesin move along microtubules?

- Towards the plus end

Which way does dynein move along microtubules?

- Towards the minus end

Describe the structures of dynein and kinesin

- 2 heads containing microtubule binding domains that walk along microtubules

- Tails bind to cargo

What are the steps in the walking of motor proteins?

1. Head 1 binds to the microtubule, head 2 binds to ADP

2. ATP binds to head 1 causing a conformational change that makes head 2 swing around

3. Once head 2 is over a binding site it binds to the microtubule and releases the ADP

4. ATP at head 1 is hydrolyzed into ADP causing head 1 to release from the microtubule

5. Process repeats

Compare and contrast the composition, movement and network formations of actin filaments and microtubules

What is the building block of actin?

- Monomeric actin protein

Describe the structure and polarity of actin filaments

- Actin monomers are bound laterally and longitudinally

- Lateral and longitudinal boding gives actin filaments a high tensile strength

- Have plus and minus ends called the barbed and pointed ends respectively

Which high energy molecule is used for actin polymerization?

- ATP/ADP

Describe the stages of actin polymerization

1. Nucleation: A third actin monomer binds to a dimer to form a nucleus trimer. Serving as the core for the rest of the actin filament to form much like a MTOC but simpler

2. Elongation: Additional actin monomers are added to the nucleus from both sides, favouring the plus end to elongate the filament

3. Rate of assembly equals the rate of disassembly and net actin filament elongation ceases

What is actin treadmilling?

- The favoured addition of monomers to one end with the same rate of monomer removal at the other end

How is treadmilling regulated?

- ATP-actin concentration compared to ADP-bound actin

- ATP-actin concentration must be lower at the plus end than the minus end

Why is treadmilling important?

- Gives cells the ability to rapidly adjust the actin cytoskeleton faster than intermediate filaments

What are actin-binding proteins?

- Proteins that bind to actin to modulate the structure, function, and disassembly of the actin cytoskeleton

What are monomer-binding proteins?

- Proteins that bind directly to the actin monomers to influence polymerization

What are nucleating proteins?

- Proteins that bind to actin polymers to increase their stability and can allow for growth of a new branch

What are capping proteins?

- Proteins that bind to the plus or minus end and can stabilize the polymer to prevent disassembly and further assembly

What are severing and depolymerization proteins?

- Proteins that can bind to the actin polymer and sever or induce disassembly respectively

What are cross-linking proteins?

- Proteins that allow the side-to-side linkage of actin polymers to form bundles of actin filaments

What are membrane anchors?

- Link filaments to nonactin structural proteins similar to integral plasma membrane proteins like integrins

What are actin-binding motor proteins?

- Proteins that bind to the actin filament and allow movement

What are myosins?

- Multi-subunit (heavy or light chain) proteins that bind to actin to allow movement

- 18 different families

Describe the structure of myosins?

- Motor domain: Formed by the heavy chain which binds to the actin filament and ATP

- Regulatory domain: Formed by a heavy chain and two light chains, moves back and forth as the myosin moves along an actin filament

- Tail domain: Binds to other cellular proteins or other myosins

How does myosin move along actin filaments?

- In an energy dependent process

- ATP bound to the motor domain is hydrolyzed to make ADP and an inorganic phosphate

- A conformational shift is caused in the regulatory domain swinging it like a lever

- Motor domain binds to the actin filament

- Inorganic phosphate is released from the myosin causing another conformational change which pulls myosin along the actin filament

- ADP is released allowing ATP to bind and cause myosin to unbind from the actin filament

Which way does myosin move along actin filaments?

- Myosin typically moves toward the plus/barbed end

How is cellular migration initiated?

- Originates within the cell itself

- Actin polymerizes near the plasma membrane and physically pushes it outwards

What are filopodia?

- Thin parallel bundles of filaments

- Polar, with plus ends facing the plasma membrane

- Extend in the direction of intended movement

What are lamellipodia?

- Sheet-like bundles of actin filaments

- Polar, with plus ends facing the plasma membrane

- Distend a wider amount of the plasma membrane in the same direction as the filopodia

What are stress fibres?

- Actin filament that are anchored to integrins

- Resemble filopodia but with different polarity, plus ends toward the cytosol

- Rich in motor proteins which allow actin filaments to move forward

What are cyclins and cyclin-dependent kinases?

- Class of proteins associated with progression through the cell cycle

- CDKs bind to their respective cyclins to become activated

- Once activated, the kinases phosphorylate other proteins to trigger the next stage of the cell cycle

What happens in the G1 phase?

- Cells are not actively dividing

- Cells are growing

What happens in the G0 phase?

- Cells are resting or quiescent

What happens in the G1/S checkpoint?

- Cell proteins check for DNA damage

- Activates signals that allows the cell to divide

What happens in the S phase?

- The cell replicates it entire genome to prep for cell division

- Centrosome is duplicated

What happens in the S/G2 checkpoint?

- DNA integrity is checked

What happens in the G2 phase?

- The last chance for cells to grow before division

- Cytoplasm amount and cellular contents are increased

What happens in the G2/M checkpoint?

- Large-scale rearrangement is triggered

- Cell volume continues to increase

What happens in the M phase?

- Mitosis occurs

What is the p53 protein?

- A tumour suppressor protein that ensures cells with damaged DNA does not divide

- Can initiate apoptosis in cells with damaged DNA

- Without it cells can evade apoptosis and replicate uncontrollably

What happens in interphase?

- Cells are not yet dividing

- DNA replication occurs

- Includes G1, S and G2 phases

What happens in prophase?

- Chromosomes are condensed and packaged into chromatids

- Each chromosome replicated to make identical sister chromatids connected at the centromere

- Gene transcription is shut down

- Endomembrane system dissolves into vesicles leaving only mitochondria intact

- Nuclear envelope dissolves

- Chromosomes released into the cytosol

- Microtubule network forms the mitotic spindle around each centrosome

What are centrosomes?

- Organelles formed by two centrioles responsible for microtubule organization

What happens in prometaphase?

- Kinetochore forms

- Chromosomes move to the centre of the cell

What is the kinetochore?

- Protein complex that binds to chromatids on either side of the centromere

- Function as molecular motors

- Use ATP to polymerize and depolymerize microtubule spindle fibres

What happens in metaphase?

- Chromosomes have arrived at the spindle equator

- Chromosomes are successfully attach to kinetochore microtubules

- Mitotic spindle checkpoint: All chromosomes must be properly aligned at the spindle equator for the next stage to start

What happens in anaphase?

- Proteins binding sister chromatids are cleaved

- Each chromosome is divided into two identical daughter chromosomes

- Kinetochore microtubules are shortened

- Chromosome. move apart towards opposite ends of the cell

- Chromosomes reach their maximum condensation level

- Microtubules organize arounf the spindle equator to develop the contractile ring

What happens in telophase?

- Rearrangements that occurred in prophase are reversed

- Nuclear membrane reforms

- Interphase cytoskeleton begins to reform

- Endomembrane system begins to reform

What happens in cytokinesis?

- A contractile ring forms at the previous mitotic spindle equator

- Ring tightens and splits the cell roughly in half

- Plasma membrane temporarily snaps but reseals spontaneously

- New cells enter interphase with normal cell architecture