Cell Bio Exam 2

Lectures 12-16

Cytoskeleton

A complex network of interlinking filaments and tubules that extend throughout the cytoplasm of the cell and is highly dynamic

Cytoskeleton Functions

1. Spatial organization of the contents of a cell that traffics organelles and divides chromosomes during division

2. Connects the components of the cell both physically and biochemically to the external environment- structural support and mechanical stress

3. Generates coordinated forces that enable the cell to move/ change shape- cell migration and chemotaxis

Cytoskeleton’s 3 Main Types of Polymers

• Microfilaments (actin)- highly organized networks of polymers and actin binding proteins scattered across the cell to respond to local signaling activity; composed of Polarized Polymers (+/-); include/relation to myosin protein motors to move/ do work; stiffness= 13.5µm

• Microtubules (tubulin)- Long tubes interacting with microtubule-associating proteins; stiffness= 5,000µm; Dynamic instability; Polarized polymers; organized by central centers (MTOC); Dynein and Kinesin protein motors; 2 types of tubulin: a and ß tubules

• Intermediate Filaments- Family of related proteins that function mainly in strengthening of the cytoskeleton; stiffness= 0.5µm; responds to mechanical stress; Non-polarized polymers; cannot support motor proteins

4Types of Microfilament (Actin) Elements/ Structures 

• Cortex- thin layer; highly cross-linked just under membrane all over the cell 

• Stress Fibers- antiparallel contractive springs that pull sides of cell in and out to get undulating movement from the cell or cilia/ flagella 

• Lamellipodia- highly branched and crosslinked networks that act like a “fist/ shock absorbers”- elastic; generates movement

• Filopodia- Parallel and cross-linked bundles that feel in the environment for pH and other 

Formation of Microfilaments

Spontaneous but highly conserved process where 3 actin monomers (an ATPase) fuse without ATP hydrolysis to undergo elongation and eventually ATP hydrolysis since only ADP- actin can be broken down; made up of two different types of subunits: ADP subunits (older part of the filament thats (-)) and ATP subunits (newer part of the filaments that (+))

Treadmilling

Phenomenon when one end of a filament grows in length while the other end shrinks at some rate resulting in a section of filament seemingly “moving” across a stratum or the cytosol 

Dynamics of Microfilament Formation

G-actin monomers are added to the (+) end/ barbed end of the filament; can be added to the (-) end but is slower.

Critical Concentration 

The concentration of subunits available at either end at which the filament will neither grow nor shrink- full stop. Usually higher concentration at (+) end because it grows more; if concentration of subunits is > that the critical concentration then growth happens, if concentration of subunits is < than critical concentration then shrinking happens 

Tip Nucleation Model

1. APC-C Dimer interacts with Formin which will pull in subunits. APC-Dimer needed when starting filament- brings Formin to structure.

2. Each Formin monomer binds to a Profilin unit attached to a G-Actin monomer. As Formin Pulls the molecules together the Profilin releases and allows subunits to fuse and build- Profilin prevents premature spontaneous generation. 

3. As filament grows, ATP released of the actin to be recycled- now older part of filament is ADP-actin. 

Arp2/3 Mediated Nucleation Model

1. Arp2/3 Complex binds to the side or (-) end of an already existing filament and is responsible for branching. 

2. Nucleation Promotion Factors deliver the G-Actin to the anchored complex and remove the Profilin. 

3. G-Actin monomers are added to the (+) end of growing filament, but Arp2/3 is anchored and cannot move with growing filament so can only build as far as it can reach before another is needed. 

ADF/ Cofilin Induced Disassembly

ADF/ cofilin binds to ADP-actin and wedges between subunits to cause them to twist, which weakens the bond leading to the depolymerization or severing; Can also remove already existing branches on a filament 

ADF/ Cofilin Induced Disassembly in Different concentrations

• In Low [ ] ADF/ Cofilin, there is persistent severing and filament is very weak- stiff with constant severing 

• In High [ ] ADF/ Cofilin, there is rapid cofilin binding which leads to slower severing rate because high ADF/ cofilin [ ] filament is flexible

Lamellipodia- Branched Networks (the Shock Absorber and fist)

• Controls cell movement and shape changes when branching networks form so fast that the fierce generated at the cell surface moves the cell. 

• Branching is formed by the Arp2/3 Complex that gets recruited by a “Primer” 

• Nucleation Promoting Factors (NPF) interacts with profilin actin and removes the profilin and puts the actin on its WH2 domain in branching 

• Branching stops when capping proteins binds to the end of actin filaments so branches stay short and defense and therefore strong

Filopodia- Parallel Actin Bundles 

• Longer filamenta produced due to lack of capping proteins; longer filaments lead to limited force produced as long branches are less dense

• Because of longer filaments, we get interaction with cross-linking proteins to help hold branches together and prevent them from falling over, without them we would lose structure and rigidity 

• No capping proteins because of Ena/VASP= an anti-capping protein 

• Ca2+ ions can control Filopodia length- High Ca concentration stop growth while high Mg2+ concentration grow Filopodia by causing them to polymerize 

Anti-Parallel Actin Bundles “Stress Fibers” 

• Contractile fibers throughout the cell consisting of unbranched actin filaments and myosin in its network underlying the inner face of the plasma membrane 

• Myosin acts as the motors to trigger the contractive movement, “springs”, which creates the undulating movement of the cell 

• Polarized

• Myosin also aides in the disassembly of the filaments 

Crosslinked Networks - “Cortex” 

Short Actin Filaments connected by cross-linker proteins, to form networks- not nucleated by Arp2/3 so therefore unbranched 

• Highly crosslinked all around perimeter of cell membrane- bound to the membrane by ERM proteins 

• Different crosslinked proteins to accomodate in size for different angles. 

• Crosslinked actin networks will adjust to outside stress applied to the cell resulting in increased elasticity- less cross-linked = more elasticity

• Can have myosin but mainly to connect the cortex to other parts of actin structure in the cell 

Myosin Induced Disassembly

• Directed motion of myosin can induce filament buckling and eventually breakage when one end of the microfilament moves faster than the other one 

• Happens at the same time as cofilin disassembly if it is an option- not one is favored 

Actin Cytoskeleton and Disease Relationship

Found that cofilin rods/ aggregates were spatially associated with neuritic plaques; the generation of cofilin rods marks a sequence of events that correlates with the development of Tau pathology and AD independent of patient age

Significance of Microtubule Reconstitution

Experiment that showed microtubules will form spontenously with very little things preventing such from happening; formed in lab from just Ca2+ chelators and GTP

Classical Polymerization Theory

Theory proposed by Oosawa on how polymers form that can be applied to microtubules, including critical concentration principle 

Critical Concentration and how applies to Microtubules

Critical concentration is the concentration of subunits free in the cell; here polymer is neither growing nor shrinking but with microtubules it is either growing OR shrinking- no in between; over all microtubule structure always maintains at least critical concentration

Dynamic Instability of Microtubules 

An entire microtubule is either growing or shrinking but it never reaches a point of stasis/ stopping 

Molecular Composition of Microtubules

Composed of a-tubulin and ß-tubulin that come together to form a dimer; Dimers form oligomer which form prototubules that that have a (+) end and a (-) end; GTP is involved but tubulin is not a GTPase, it is just a G-Protein

GTP in Tubulin binding

a-Tubulin binds to GTP, ß-Tubulin then sits on top and keeps the GTP from being hydrolyzed (like a cap). If GTP binds to ß-tubulin it is exposed and will hydrolyze creatign GDP

GDP allows for bending of the oligomer and eventually catastrophe. GTP provides structure and rigidity to the oligomer. Leads to rescue. 

Intra-prototubule Contacts 

Interactions going up the prototubule- a and ß interactions that are part of one prototubule and hydrophobic

Inter-prototubule Interactions

Interaction of prototubules laterally→ a-a and ß-ß interactions spanning across multiple prototubules that are hydrophilic; Inter-interaction of 13 (usually) prototubules leads to folding and the formation of a microtubule

3-Start-Helix 

Structure of newly formed microtubule that has some misalignment creating a seam of non-like subunits; the not flat top helps with attachment to the gamma complex for the sake of nucleation

GTP Cap

A GTP cap composed of a-tubulin bound to GTP and ß-tubulin bound to GTP dimer is always added to the (+) end of the growing microtubule 

GTP Islands

Regardless of if a microtubule is growing or shrinking, there will be random spots of GTP- bound ß-tubulin that gets protected/ remains unhydrolyzed since only GTP bound tubulin can bind to another GTP so these islands allow for rescue to happen later after catastrophe. The islands mark where catastrophe will stop.

Catastrophe

The transition from growth to shri