Lecture 13 Flashcards
How do things move?
1) Cytoskeleton as tracks for motor proteins
2) Cytoskeleton for movement (actin filaments, microtubules)
3) Brownian Motion
What are the 2 cytoskeletal elements that are used as track because they are POLAR filaments?
Actin filaments (Myosin)
Microtubules (Kinesis, Dynein)
What is most intracellular movement in ANIMAL CELLS due to?
It is due to material moving along microtubules
What is most intracellular movement in PLANTS and FUNGI due to?
It is due to material moving along actin filaments
What do all molecular motors have in common?
Use ATP for their movement
Have 2 parts (a motor domain [Head] and a tail), they are usually linked together by a neck (AKA. neck-linker)
What is a motor domain?
Uses ATP hydrolysis to move the motor
What is a tail?
Binds to cargo and can bind to other parts of the motor to inhibit the motors function when not in use
What will happen to the cytoskeletal element if a motor is anchored in position?
Then the cytoskeletal element will physically move when the motor is activated
What will happen to the motor if the cytoskeletal element is anchored in position?
Then the motor will physically move when the motor is activated
What are the only molecular motors that use actin as tracks?
Myosin
Which way does myosin move?
Usually moves towards the barbed end (+ end) of actin filaments… only 1 moves towards the - (pointed end)
What does myosin use for movement?
An ATPase. Uses ATP for movement
How many heads can myosin have?
Can have 1 or 2 heads, which are the motor domains
What are the heavy chain components of the head?
1) Binds and hydrolyzes ATP
2) Binds to Actin filaments
What are the light chain components of the head?
There are about 1 to 7 of them depending on the myosin
Activate the myosin in the presence of Ca2+
What does myosin attach to?
It attaches to many types of tails
Where were myosins discovered in?
They were discovered in skeletal muscle
What do muscle myosins do?
It creates the force for skeletal muscle contraction
How many binding sites are activated during normal muscle contraction?
Only 10-20%
What is muscle myosin called?
Myosin II
How many heads does Myosin have?
Has 2 heads attached to a long coiled-coli tail that intertwines.
These are called the thick filaments (in skeletal muscle).
The 2 strands of actin filaments that they bind to are called the thin filaments.
What terminal of amino acids forms the globular catalytic domain?
The N-terminal 710 amino acids form the globular catalytic domain.
Where does ATP dock into?
ATP docks into the nucleotide binding site of the molecule
Where does Actin bind?
Actin binds ~4 nm away from the ATP on the other side of the head
What does the globular head bind to?
The globular heads bind tightly to actin in the ABSENSE of ATP. This causes rigor mortis (no ATP produced when dead). Nearly 100% of binding sites are triggered during rigor. The heads only release in the presence of ATP.
What happens when heads are isolated?
When heads are isolated and used for actin experiments they bind to the filaments forming arrow-head shapes. This indicated the pointed and barbed ends of the actin filament.
What does myosin bind to?
Myosin binds to 2 adjacent actin subunits within the actin filaments
What are sarcomeres?
The structural unit of actin (thin filaments) and myosin (thick filaments) in skeletal muscles are called sarcomere
Are there also cytoplasmic Myosin II’s in the general class of Myosin II proteins?
Yes
What are thick filaments called?
Muscles (Myosin II)
What are thin filaments?
Actin filaments
How many classes are there are the Myosin superfamily?
35 classes (so far in eukaryotes)
How many motors are “orphans” that don’t fall into those classes?
145
How many organisms have the genes from all classes?
No organism has the genes from all classes, onlt a subset of classes
How many myosin genes and classes do humans have?
Humans have 40 myosin genes from 13 classes
Where did Myosin originate from?
All originated from a gene similar to Myosin I, but plants have lost Myosin 1.
What did that gene then originate?
The gene then originated the Myosin V gene
What do diverse tails give?
Diverse tails give myosins their specificites for different cargo
What are some ways that myosin got their diversity?
Gene duplication
Acquired extra domains
Evolutionary Divergence (Got divergent tails to bind to different cargos)
What is the first unconventional myosin discovered?
Myosin 1
What are heavy chains encoded by in Myosin 1?
8 genes
What Myosin group has a very diverse class of Myosins, large in number?
Myosin 1
How many heads does Myosin 1 have?
Only have 1 head, so many must work together for movement, but 1 must always remian attached to the actin filament so they don’t fall off
How many light chains are in Myosin 1?
Variable number of light chains can associate with them
Is there a head and tails association in Myosin 1?
The heads don’t really associate with the tials, so they have very short tails.
What kind of domains do Myosin 1 have?
Have odd domains including:
1) A basic domain that binds to acidic phospholipids
2) SH3 (src homology-3) domain to bind to proline-rich areas of other proteins
What kind of domains do Myosin 1 have?
Have odd domains including:
1) A basic domain that binds to acidic phospholipids
2) SH3 (src homology-3) domain to bind to proline-rich areas of other proteins
What engulfing process is Myosin 1 involved in?
Involved in endocytosis (it is normally concentrated at sites of phagocytosis and macropinocytosis)
How does Myosin 1 function in linking two things together?
It links the actin of microvilli to the membrane
What is the structure of the chain in Myosin V?
Has a very long light chain domain
1) This allows it to take very long (36 nm) steps along the actin filaments
2) When only 1 head was labeled with a fluorescent probe, 72 nm movements were detected
What does Myosin V transport?
Pigment granules, ribonucleic proteins, mRNAS, enzymes
What are the most processive motors to date?
Myosin V. It can take multiple steps without falling off of the actin filaments).
One head binds before the other releases.
What does the gene for Myosin V give rise to?
The gene for Myosin V gave rise to Myosins VIII and IX
Do Myosins V, VIII, and IX move slow or fast?
They move very fast
What do defects in Myosin V cause?
Seizures
Gricelli’s syndrome (a recessive human disease of pigmentation dilution)
Immunodeficiencies
What happens when the Myosin V motor is not in use?
When not in use the motor folds up on itself. The tail interacts with the head
What is the only myosin to move to the pointed (-) ends of actin filaments?
Myosin VI
What causes this movement in Myosin VI?
Caused by the lever arm naturally swinging in the opposite direction as other myosins. Myosin VI is an actin-based motor that moves backwards.
Is Myosin VI a monomer?
It is a monomer, but adaptor proteins can dimerize it, allowing for large 30nm steps.
How does Myosin VI relate to vesicles?
Can move vesicales from the plasma membrane into the cytoplasm during endocytosis.
What do mutations in human Myosin VIIa (7a) cause?
Mutations cause deafness at birth, then retinal degeneration, which leads to blindness
Is Myosin VIIa a monomer?
It is a monomer, but can dimerize. When dimerized in vitro and in vitro, it is processive but it is very slow. This hasn’t been shown in vivo yet.
Are there cargo receptors in Myosin VIIa?
No cargo receptors have been identified
What is the function of Myosin VIIa?
Unknown, but it moves so slowly that some researchers think it might act as a structural myosin, bundling actin filaments together.
Others think it could act as a molecular force sensor.
(It would stall when forces became too strong when moving along actin filaments)
What is Myosin 18A?
Has multiple spliced forms and differing subcellular localizations
How is Myosin 18A similar to Myosin II?
It’s core (coiled coil domain) is most similar to Myosin II, but it is still quite divergent.
What is the IQ domain in Myosin 18A?
IQ domain: where the light chains bind (very small domain)
What is the PDZ domain in Myosin 18A?
Has an amino-terminal PDZ domain (usually used for protein protein interactions). This is unique to this myosin. No other myosins have PDZ domains (large domain)
What is the KE motif in Myosin 18A?
KE motif: Lysine-Glutamic acid repeat region (unknown function)
What do many post-translational modifications get in Myosin 18A and actin?
Gets Tyrosine, Serine, Threonine phosphorylated, ubiquitylated, acetylated, methylated (largely based on proteomics screens). These modifications have unknown functions on the protein
What is Myosin 18A and actin involved in?
Is involved in the retrograde treadmilling of actin. Has been found as part of protein complexes
What did immunoprecipitations find in the binding in a complex in Myosin 18A and actin?
Immunoprecipitations found MYO18A Myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) and Leucine-rich adaptor protein 1 (LURAP1) binding in a complex
What does LURAP link in Myosin 18A?
LURAP links MRCK to MYO18A
What can MRCK do to MYO18A?
MRCK can phosphorylate MYO18A
What does MRCK phosphorylating MYO18A lead to?
1) This leads to increased cellular protrusions
2) This complex is involved in the retrograde flow of the actin-myosin complexes at lamellipodia and the center of the cell
Is Myosin 18A a motor?
Myosins usually bind to actin through their motor domains. There is no interaction of the Myosin 18A motor domain and actin, even with ATP depleted (Note: ATP depletion causes very strong binding of myosins to actin in other systems)
Is there a connection between MYO18A and actin?
MYO18A and actin do bind between the KE and PDZ domains at the N-terminus.
What cannot hydrolyze ATP at all?
Drosophola Myosin 18A (called Mhcl, Myosin heavy chain-like) cannot hydrolyze ATP at all
What can hydrolyze ATP weakly?
Mouse Myosin 18A can only weakly hydrolyze ATP from its light chains and binds only weakly to actin.
What is the target and mechanism of blebbistatin?
Target: Myosin II
Mechanism: Hinders Pi release
What is the target and mechanism of BTS?
Target: Myosin II
Mechanism: Hinders Pi release and ADP release
What is the target and mechanism of BDM?
Target: Myosin II
Mechanism: Hinders Pi release
What is the target and mechanism of PCIP?
Target: Myosin I
Mechanism: Reduces coupling between actin and nucleotide binding sites
What is the target and mechanism of PBP?
Target: Myosin V
Mechanism: Reduces ADP dissociation, ATP binding/hydrolysis, and coupling between actin, nucleotide binding sites.
How often is the cell cortex under tension?
The cell cortex is ALWAYS under tension
What generates the tension in the cell cortex?
Non-muscle myosin-2 genereates the most cortical contractile tension
How does non-muscle myosin-2 generate the most cortical contractile tension?
Through pulling on actin filaments at the cell cortex, this leads to stress in the cortical actin network, which then leads to stress at the membrane. Myosin-2 does this by generating bipolar minifilaments at the cell cortex
How many heavy chain isoforms are there?
There are 3 non-muscle myosin-2 heavy chain isoforms
What do all 3 non-muscle myosin-2 heavy chain isoforms localize to?
They all localize to the cell cortex (Myosin-2A, -2B, -2C)
What do these isoforms do?
These isoforms can co-assemble into mixed minifilamemts too
What does Myosin-1 do?
Myosin-1 also binds actin to the membrane at the cell cortex as does Myosin-18. These can all co-assemble with non-myscle Myosin-2 at the cortex.
