Premed Adventurer

Non-Muscle actin filaments are abundant in eukaryotic cells

Microvilli

Bush boder epithelia of intestine

They increase surface area

Are hair cells of the inner ear (sound detection)

Stress fibers

Adhesion and Cell Shape (Fibroblasts growing in in vitro)

Filopodia and Lamellipodia

At the leading edge of the moving cells 

Contractile Ring

Division of cytoplasm in animal cells

Cortical Actin

Just beneath plasma membrane of most Euks, anchors membrane

Intracellular Transport

Myosin-coated organelles moving along actin filaments in plants

Actin polypeptice assembles in microfilament (F-Actin)

ATP affects assembly dynamics

G- Actin

Tertiary structure

F- Actin

Quaternary structure

Actin filaments are polar. They are added at the plus end and are taken out at the minus end

Two-Stranded Helix shape

Actin Filaments in cells are often Dynamic

5% of cell protein is actin and about half is polymerized filaments

**** ATP binding to actin affects dynamics

C-actin with ATP bound adds + end filaments preferentially grow at plus end

Away from plus end, ATP is hydrolyzed. This induces conformational change in actin subunits.

ACTIN IS AN ATPASE

ADP actin subunits do not bind each other as strongly

G-actin with ADP bound is preferentially released at – end, shortens filament

DYNAMICS ALSO REGULATED BY ACTIN-BINDING PROTIENS

Filaments can grow and shorten rapidly

Treadmilling

This is the same addition to plus and subtraction at minus we just discussed

This is how the actin filaments crawl through the cell

Filaments move left to right but subunits don’t move

Dynamic F-actin can disassemble at one location ad reassemble elsewhere

Actin depolymerizes at sperm entry site and repolymerizes at new rhizoid pole

Actin marking the rhizoid pole is dynamic

Dynamic Actin at the surface of a moving cell

Lamellipodia

Ruffles- sheet-like extensions of the cells leading edge

Filopodia

Microspikes- finger-like

Motility is dependent upon actin assembly

Arrangment of actin filaments in lamellipodia and filopodia

Stress fibers

Contractile bundle

Antiparallel

Lamellipodium

Branched network

Filopodium

Parallel filaments

Actin assembly drives extension of lamellipodia and filopodia

Modulating the assembly/function of actin:actin-binding proteins

Regulate structure of F-actin array, and dynamics of array

Nucleating proteins

Crosslinked proteins

 Bundling protiens

Motors

ARP 2/3 Complex (Nucleating Proteins)

In Euks

Nucleation of actin filaments from pure subunits is very slow

Actin nucleation-related proteins (ARPs 2 and 3)

And several other polypeptide in the macromolecular complex

ARP2/3 “nucleates” actin filament assembly by providing a template or “seed” that can be elongated by subunit addition

The ARP 2/3 complex caps minus-end of actin filaments

Capped ends are less dynamic

ARP 2/3 nucleates branching network in lamellipodia

Activated ARP 2/3 binds to side of existing actin filaments

And nucleates new filaments from the side (branches)

New, elongating filaments are not yet capped and push leading edge outward

Network depolymerizes in rear

COMMON IN ALL EUKS SO FAR

Actin filament assembly drives extension of lamellipodia

Actin filament assembly drives forward membrafe extension

Actin filaments disassemble behind leading edge

Filament Capped when finished elongating

Actin movement also drives movement of intracellular parasites

Initiation of actin assembly is regulated by Rho family GTPases

Part of Ras superfamily of G proteins that serve as molecular switches

Allow rapid assembly and disassembly of actin-based structures in response to enviro cues

Rho activation (GTP bound confirmation) causes formation of ‘stress fibers’

Rac (Rho Subfamily) activation causes formation of massive lamellipodium

Cdc42 (Rho Subfamily) Activation causes formation of filapodia

The Rho GTPase triggers activation of ARP2/3 complex (but form different arrays)

Bundling vs. Cross-linking: a-actin vs. filamin

Bundling

A-actin (dimer) is rod-shaped with two actin binding sites:

Forms loose parallel bundles of actin filaments

Z-line of striated muscle

Stress fibers and focal contacts

Filopodia

Cross-linking

Filamin (dimer) has actin binding sites on long flexible arms

Forms cross-linked actin gels

Stress fibers and focal adhesion

Smooth muscle

Actin filament bundles called “stress fibers” are common in cultured fibroblasts in animals

Stress fibers

                                 i.     Provide mechanical strength to cell

                               ii.     Attach cell to substratum at focal point of adhesions

Polarity of actin filaments in bundle is not uniform

Organized by actin binding proteins

Bundling (a actin)

Crosslinking (filamin)

Type II myosin

How do stress fibers attach to substratum?

Integrins

Integrins link stress fibers to extracellular matrix at ‘focal adhesions’

Integrins bound to linker proteins which are bound in actin filaments

Defects in integrins can cause muscular dystophe

Extracellular matrix is composed of secreted proteins and glycoproteins

Linkage from stress fiber to ECM attaches cell to substratum at focal adhesion

There are many other linkages from actin to ECM in animal cells

Actin filaments in plants are also linked to the ECM (cell wall) but are linking molecules are different

Non-muscle cells contain multiple myosins

17 subfamilies of myosins- gene duplication and divergence

Note** that some have 2 heads and some only have one

Myosin II = 2 headed, discovered first in muscle, also in non-muscle cells

N terminus- motor domain of heavy chains

Coiled-coil tail

C-terminus of heavy chains

Each head is independent of the other

Myosin is an ‘actin dependent’ ATPase that acts as a molecular motor

No nucleotide. Myosin head is tightly bound to actin

ATP binding releases myosin from actin

                                 i.     ATP binding site changes the conformation of myosin

ATP hydrolysis “cocks” myosin, myosin binds weakly to actin

Pi is released, strengthens binding of myosin to actin

Myosin bound tightly to actin undergoes “power stroke” and then ADP is released

                                 i.     Myosin head walks towards the plus end of the actin filament

                               ii.     Mechanism is the same for muscle and non-muscle actin

Non-muscle myosins perform many roles

“type II myosin:” slides antiparallel actin filaments

                                 i.     Myosin walks towards the plus end

                               ii.     Cell contraction and cell division

Single headed myosins

Vessicle transport –cytoplasmic streaming inalgae and plants

Links actin to membrane – Minus end leading. Myosin walks towards plus end

Myosins coat organelles and power cytoplasmic streaming in green algae and plants

-       Stirs the cytoplasm, plant cells are often too large to rely on simple diffusion

-       Myosins power cytoplasmic streaming in green algae and plants

-       Vesicles, ER, Nucleus, and other organelles move along actin cables on subcortical cytoplasm

-       Powered by myosin 6 speeds ip to 7 micrometers per second

Myosins and cell motility

-       Type 2 mosins found in the tail (contractile)

-       Type 1 founding in leading edge

Myosins work in conjunction with actin polymerization to drive movement

A model for motility using actin and myosin motors

-       Focal adhesion

-       Actin polymerization extends lamellipodium (myosin 1)

          - Focal adhesion and actin disassembly

- Contraction of myosin II

Neutrophils are chemotactic- movement towards or away from chemical

   -Change direction is mediated by activation of RHO GTPases

   - Crawl towards N- formyl peptides

  Cytoplasmic 

Keratins- these are epithelial. So in your hair. Skin. Nails. Claws.

Vimentin- connective tissue, muscle cells, neuroglial cells 

Neurofilaments- Nerve cells 

Nuclear 

Nuclear lamins- in animal cells 

Characteristics-

~The are located between actins and myosins in the muscles 

~They have a strong rope like structure, gives shape to nucleus euks and cytoplasm of vertebrate cells 

~Provides Tensile Strength that allows layers of cells to stretch without rupture. (Resists mechanical stress)

Keratins Protect Skin Cells exposed to mechanical stress

Basal cells express keratins k5 and k14

If there is a point mutation in these it disrupts keratin filaments

So it causes things like epidermolysis bullosa simplex

Other ailments

Neurofilaments: ALS

Lamins: Muscular dystrophy and premature aging

Let's talk about cell surface receptors.  The ligands for these are hydrophobic. This makes sense because they are not polar and therefore cannot diffuse into the cell.  The receptors for these are located on the cell surface.  the ligand binds to the receptor and and intracellular signal is sent out. this signal goes into the nucleus. In the nucleus the signal gives the orders to translate and transcribe this signal.