Actin and IF

Building Blocks of MF

• MF are the smallest filaments

• MF is F-actin (filament)

  • Best known for muscle contraction

• Play a role in cell migration, amoeboid movement and cytoplasmic streamin

Actin

• Highly conserved globular protein

• Alpha actin is muscle-specific

• Beta and gamma is non-muscle actin

  • Beta actin is on the apical side and gamma actin on the basal side in epithelial cells

• Also forms stress fibres and a contractile ring during cytokinesis

• Once synthesized it folds into a globular U-shape molecule that can bind ATP or ADP

• G-actin polymerizes to form F-actin, the microfilaments

• MF hydrolyzes ATP

Structure of ACTIN

• G-actin can polymerize reversibly into filaments with a lag and elongation phase

• F-actin is composed of 2 linear strands of G-actin wound into a helix

  • 13.5 monomers per half-turn

• All the actin monomers in the filament have the same orientation giving the strand polarity

  • Each end is structurally and chemically different

• Organized into many bundles and networks

• Dispersed throughout the cell but concentrated underneath the plasma membrane

• Very dynamic, able to assembly/disassemble itself

• Requires ATP, Actin is ATPase

Use of MF in the Cell

 

Cell can regulate where/how MF are assembled, generating many structures

• Cells that crawl have lamellipodia and filopodia at their leading edge

• Cells that adhere tightly to an underlying substrate have an organized bundles called stress fibres

What the plus and minus end refer to

• Plus end is called Barbed End

• Minus end is called Pointed End

Role of Nucleotides in Actin polymerization

• Faster addition of G-action at the plus end than the minus end

• Once G-actin monomers assemble onto MF, the ATP that is bound to it slowly hydrolyzes

  • Hydrolysis isn't necessary for polymerization

• Growing ends of MF have ATP-actin

• Most of Actin is composed of ADP-actin

In vitro nucleation/elongation

• Critical concentration of plus end is 0.2 micro molar

• Critical concentration of minus end is 0.4 micro molar

• Plus end adds actin faster

In vivo Actin Assembly

• Actin filament assembly is controlled by formin proteins and the ARP2/3 complex

• Formin

  • Control the assembly of actin filaments in vivo

  • It is a large multi-domain proteins that directly nucleate polymerization of unbranched actin filaments

  • Remains associated on the barbed end, rapidly inserting acti subunits and protecting it from capping proteins

• Arp2/3 complex nucleate new filaments

  • Becomes activated when Arp2/3 combines with nucleation-promoting factors

  • When activated it resembles the plus end of an actin filament allows actin subunits to assemble on the structure and bypass nucleation (slowest step in MF formation)

  • It nucleates new branches on the sides of MF

  • Arp2/3 forms a template to which actin monomers can be added

  • Arp2/3 complex Is associated with structures at the leading edge of migrating cells

    • Localized in regions of rapid actin filament growth like lamellopodia

Function of Actin-binding proteins

• iolate monomers to regulate rate of polymerization

  • Availability of ATP-bound G-actin will effect MF assembly

• Actin-binding proteins that can cap actin filaments

  • Capping proteins binds to the end of a filament to prevent further loss of addition of subunits; this can stabilize MF

• Actin-binding proteins can sever Actin filaments

  • MF networks can be broken up by roteins that bind to the side of a MF and break it into two

• Actin-binding proteins can bundle actin filaments

  • Some actin-containing structures are highly ordered, enhancing rigidity of the structure

  • Actin may be bundles into tightly organized arrays called focal contacts or focal adhesion

  • Fimbrin bundle MF in microvili

•  Actin-binding protein can link MF to membranes

  • MF can connect to the plasma membrane to exert force, protruding outwards or invaginating inward

  • Indirect connections to the membrane requires one or more peripheral membrane proteins

Thymosin Beta 4

Binds ATP-actin monomers and prevents them from polymerizing

Profilin

• Binds to ADP G-actin and catalyzes the exchange of ADP for ATP, promotes polymerization

Cofilin

• Binds ADP-actin in a MF

  • Severing it and promotes depolymerization

CapZ

Binds to plus end to prevent addition/loss of subunits there

Tropomodulins

Binds to minus ends, prevents addition/loss of subunits there

Gelsolin

Breaks actin MFs and caps newly exposed plus ends, prevent further polymerization

Cofilin

A filament-severing protein, facilitates depolymerization

Filamin

• Acts to join two microfilaments together where they intersect

• Has 2 actin binding sites

Fimbrin

 bundle MF in microvili

Sceptrin and ankyrin

link membrane protein to MF

Role of Intermediate Filaments

• Most Stable and least soluble

• NOT polarized

• An abundant IF is Keratin, important component of structures that grow from skin in animals

• IF support cytoskeleton by acting as a scaffold

  • Bridges molecules

  • Plectin connects different components

• Dynamically remodel

  • Nuclear lamina on the inner surface of the nuclear envelope disassemble at the onset of mitosis and reassemble after

Nuclear Lamina

• Dense fibrous network located just inside the inner nuclear membrane

• Composed of lamin protein which are IF proteins (lamin A, B, C)

• Provides mechanical support ot the nuclear envelope

• Organize nuclear pore

• Play a role in chromatin organization

• Involve in nuclear disassembly/reassembly during mitosis

Using IF to diagnose certain Tumors

• Tumor cell lose their normal morphological appearance but retain normal complement of cytoskeletal protein

• Can use antibodies against IF proteins and can determine if the origin of a tumor is epithelial, mesenchymal or neuronal tissue

• EX malignant breast/GI tract tumor contains keratin then tumor originated in epithelial

Type of proteins that comprise IF and organization

• All have a central rod-shaped alpha helix domain that is conserved in size, secondary structure and sequence

• The N-term/C-term domains differ greatly among IF proteins

• IF is made of up to 70 different protein

• IF only in animals

  • Expression varies from tissue-to-tissue

• IF are encoded by a large gene family and classified based on degree of amino acid related ness

• 5 groups

• N/C-term sequences and structures vary the most as it has binding sties for IF, MF, MT

How does IF differ from Actin and MT

• All have a central rod-shaped alpha helix domain that is conserved in size, secondary structure and sequence

• The N-term/C-term domains differ greatly among IF proteins

• Most Stable and least soluble

• NOT polarizared

IF Assembly

1. IF proteins are primarily fibrous molecules with a globular domain on each end

2. 2 polypeptides spontaneously interact and wrap around each other creating a rope-like dimer (coiled-coil)

3. 2 dimers assemble with C/N terminal in an anti-parallel orientation, causes no polarity

   1. Creates a tetramer

4. 8 tetramers pack laterally together to form a filament that is 1 unit long 

5. Units associate with each other to form elongated IF

   1. Does not require energy from either ATP or GTP