Chapter 9.2 - Actin and intermediate filaments

intermediate filaments

strong flexible ropelike fibers that provide mechanical support to cells that are subjected to stress

what cells are intermediate filaments found in

animal cells

how are intermediate filaments different from actin filaments and microtubules

chemically heterogenous group of structures that are encoded by approximately 70 genes different genes

how are IFs divided into classes

based on the type of cell in which they are found (as well as biochemical, genetic, and immunologic criteria

t or f - IF assembly does not involve ATP or GTP hydrolysis

true

structure of IFs in the cell

radiate through the cytoplasm of a wide variety of animal cells and are often interconnected to other cytoskeletal filaments by thin, wispy cross bridges

what do many IF cross bridges contain

elongated dimeric protein called plectin

plectin binding sites

has a binding site for an intermediate filament at one end and depending on the isoform a binding site for another intermediate filament, microfilament, or microtubule at the other end

basic building block of IF assembly

thought to be a rodlike tetramer

how do the tetramers of IFs assemble

eight tetramers associate with one another in a lateral arrangement to form a filament that is one unit in length

do IFs have polarity

no, its tetrameric subunits do not have polarity which distinguishes it from IFs from other cytoskeletal elements

IF solubility compared to other cytoskeletal elements

less sensitive to chemical agents than other types of cytoskeletal elements and more difficult to solubilize

what happen when labelled keratin subunits are injected cells

rapidly incorporate into existing IFs, not incorporated at the ends of the filament but into the filaments interior

how are IFs assembly and disassembly controlled

controlled promarily by subunit phosphorylation and dephosphorylation

keratins are what type of cytoskeletal element

IFs

what residue do keratins tend to have a lot of

cysteine

most diverse IF family

keratins

three major components of the nuclear envelope

• nuclear pores

• nuclear membranes

• nuclear lamina

what happens to the nuclear envelope at the end of prohase

disassembles

what is the nuclear lamina composed og

intermediate filaments (lamin) and membrane associated proteins

how are lamina disassembled

phosphorylated which causes de-polymerization and subsequent disassembly of the lamina

epidermolysis bulliosa simplex (EBS) arises from what

mutations in a gene that encodes a keratin polypeptide

desmin related myopathy

mutations in the gene that encodes desmin

what does desmin do

integrates the various components of a muscle cell

what does desmin related myopathy lead to

skeletal muscle weakness, cardiac arrythmias, and eventual congestive heart failure

functions of microfilaments (actin)

• motility

• shape

• structural support

• muscle contraction

most abundant protein in cells

actin

g actin

globular

f actin

filaments

what happens to actin monomers in the prescnce of ATP

polymerize into a flexible helical framework

minus end of actin

the end of the filament with an exposed binding cleft, binds plus end in a filament

plus end of actin

the other end is where the minus end of G actin binds

pointed end

minus end of actin

barbed end

plus end of actin

how is the ATP binding cleft oriented in actin

in the same direction in all actin subunits (monomers) in the filament

in vitro polymerization of lus and minus ends of actin

have different polymerization rates

in vivo polymerization rates of the plus and minus ends of actin

polymerization only ocurs in the plus end

why does tee plus end only polymerize in vivo for actin

minus end MAY be anchored

what dissociates in actin

only ADP-actin dissociates

how is actin polymerization and organization regulated

actin binding proteins

what dies actin bind to a lot in eukaryotic cells

many many acessory proteins

what are myosins (type of proteins)

actin based motor porteins

what do all mysoins have in common

have a characterituc head or motor domain (ATP-ase activity)

how are myosins goruped

grouped into conventional and unconventional myosins

what direction to myosins move

to positive end except myosin VI

conventional myosins

type II

what are type II myosins composed of

6 polypeptide chains

• ine pair of heavy chains

• two pairs of light chains

symmetry of type II mysoins

highly asymmetric protein

mysoin II consists of

• a pair of globular heads that contain the catalytic site of the molecule

• a pair of necks, each consisting of a single, uninterrupted α helix and two associated light chains

• a single, long, rod-shaped tail formed by the intertwining of long a-helical sections of the two heavy chains

in what way does myosin II assemble

into fibers with the ends of the tails pointing toward the center and the globular heads pointing away

skeletal muscle is used for

voluntary movement

muscle

bundles fo parallel muscle fibers (cells) joined by tendons to the bones that the muscle must move

fibers

each fiber is a multinucleate cell formed during embryogenesis and specialized for contraction

myofibril

thinner cylindrical strands that make up a muscle fiber and consist of repeating units of sacromeres

sacromeres

the contractile unit of myofibrils each of which hs a very specific organization

organization of sacromeres

each sacromere extends from one Z line to the next Z line

thin filament

actin

thick filament

mysoin

actin orientation

plus ends anchored at Z lines

capZ

caps actin at the plus end

tropomoulin

caps the minus end and regulates the length of actin filaments

nebulin

repeating actin binding motifs that binds actin filament to Z line

myomesin

bundles of the myosin filaments

titin

extends through the myosin filaments (thick) and attaches to the Z line - helps prevent tearing of muscle

sliding filament model of muscle contraction

• During contraction, the myosin molecules pull the surrounding thin filaments (actin), forcing them to slide toward the center of the sarcomere

• Individual myosins work asynchronously, so that only a fraction are active at any given instant

• The “neck” acts as a lever, amplifying the conformational change caused by ATP hydrolysis

contrctile cycle (what is it)

Conformational changes (mechanical) in the myosin head couple ATP hydrolysis (chemical) to movement

contractile cycle steps

• ATP binds to the cleft in the myosin head, releasing myosin from actin

• ATP hydrolysis to ADP+Pi causes weak binding to actin

• Pi release causes tighter binding and the power stroke that moves the thin filament toward the center of the sarcomere

• ADP is released, freeing the ATP binding cleft 1) ATP binds to the cleft in the myosin head, releasing myosin

what do calcium ions trigger and how

contraction via troponin and tropomyosin

tropomyosin

masks the myosin binding sites on the actin filament

troponin complex has how many subunits

3

troponin complex

binds to tropomyosin

role of tropomyosin and troponin

both have regulatory roles in contraction

calcium binding to troponin ____

relieves th etropomyosin blockages of the interaction between actin and myosin head

steps of contraction due to calcium

• Motor neuron excitation signal

• Signal transduction pathway leads to Ca2+ release from the SR

• Ca2+ binds to troponin (TnC subunit), causing conformation shift

• Troponin conformation shift moves tropomyosin out of place

• Myosin binding site on actin is exposed

• When excitation signaling ceases, Ca2+ are pumped back into SR, muscle relaxes

Familial hypertrophic cardiomyopathy:

Genetically dominant inherited mutation in myosin (~2 per 1000 people)

Familial hypertrophic cardiomyopath can cause

Over 40 different point mutations can lead to:

• Heart enlargement

• Abnormally small coronary vessels

• Cardiac arrhythmias

cofilin

binds ADP actin and severs filaments promoting depolymerization

profilin

• functions as an adenine nucleotide exchange factor

• Binds to ADP actin (at the plus end), changing the conformation and allowing binding of ATP

• Binding results in dissociation of profilin

• ATP actin then either joins a growing filament at the plus end OR is bound by:

thymosin

• sequesters G- actin preventing polymerization

• Displacement of thymosin allows binding of G-actin to the plus end

whta deos cpping do in F actin

stabilizes it

what does capping the plus end do in f actin

prevents further growth

what does capping the minus end do in f actin

prevents to loss of subunits

in muscle, which en of f actin is capped

both, prevents the loss or gain of subunits this way

what can actin be lined to

other actin filaments or indirectly to the cell membrane

cell cortex

network of actin filaments and accessory proteins that underlies the plasma membrane in most eukaryotic cells

Arp2/3 complex

actin related proteins, nucleates new branches off the sides of existing filaments

what are Arps activated by

WASPs (Wiskott-Aldrich syndrome protein)

to what end is g actin added

plus end

how can filaments move the cell membrane forwards

• 1 and 2) Extracellular signal recognized and signal transduction cascade initiated

• 3 and 4) WASP activates Arp complex and Arp nucleate new actin filaments

• 5) result is branch formation at plus end

• 6) Membrane is pushed forward

• 7) Caps terminate elongation

• 8) Oldest part of filament at the minus end

• 9) F-actin servered and depolymerized at minus end

• 10) Profilin exchanges ADP to ATP

what regulates the actin skeleton

Rho family of small GTPases

where is Rho bound and what does that do

• Rho family GTPases often are bound to a guanine nucleotide dissociation inhibitor (GDI) in the cytosol

• The GDI prevents Rho from interacting with it’s GEF at the plasma membrane