SPUDICH, Jim MYOGLOBIN Molec Motors Jan 17 2012

editor of DELL,  editor of PNAS...  

aspect ratio of his slides are stretched  on this HDTV screen..

The myosin family of molecular motors...

The myosin superfamily at  a glance... slide 1

myosin carries out golgi functions,  exocytosis, endocytosis

40 difft myosins in every cell in the body...

filopodium... full of actin myosin... gives them rigidity...

myosin... 2 headed molecules

when ATP binds to the head... it swings forward... 70 degr stroke that pulls
the back leg forward...

it walks across  an actin filament  by taking 25 steps or so...

Kron and Spudich  PNAS1986...

label them with fluor...

they move at 5 microns per second...

just purified actin and myosin and ATP.. that's all u need...

use laser beams to trap  polystyrene beads and glue those onto actin filament.

see Simmons, Finer  1993  Adv Exp Med Biol

or  Finer, Simmons, Spudich  Nature 1994...

put  one myosin on top of a bead that is walking on actin...

muscle myosin produces  nanometer displacements and pNewton forces...

this was in rabbit skeletal myosin...

can get faster velocity by tuning attachment time;
some are binding while others are letting go..

stroke size = 10 nano..
force is abt 5 picoNewtons...

do this expt with myosin V...

a single myosin V molecule is processive and takes 36 nm steps along actin

SHREC shows Myosin V stepping head over head... each step is 36 nano.

see it with  fluor...

Dunn and Spudich, Nature Struc and Mol Biol  2007...

attach gold particle to back leg...

gold bead steps forward... can see it on darkfield microscope..

All of this applied to myosin V strongly support the lever arm hypothesis...
lever arm swings 70 degrees pulling the rear head forward..

***
On to myosin VI...

walks in the opposite direction

V move stow ard + end of actin

VI moves  toward the - end..
myosin V has 36 nm steps...

but look at myosin VI  looks tiny,  like a dwarf... tiny legs  and yet
it also takes 36 nm steps... what gives???

short constructs move toward the + end, like myosin II and myosin VI;

Myosin VI stepping can be explained by a lever arm swinging  180 degrees.

lever arm rotation is really large... that's why  it can step so far...

Ann Houdouse  worked out the crystal struc = 1.75 angstrom...  fits perfectly...

myosin 6  swings 180 degree and has  12 nm long single apla helices  bridging the gap.

this lever arm  corroborated the lever arm hypothesis...

******
Hypertrophic and dilated cardiomyopathy...

HCM = hypertrophic cardiomyopathy... and 
dcm = dilated cardiomyopathy

missense mutation... 1 in 500 people carry it..

many of the missense mutaitons are in beta cardiac myosin itslef !!

no one had been able to express human beta-cardiac myosin in a functional form...

but it was synthesized  and sent to jim...

****

Kathy Ruppel... super star md , phd student... did much of the work that jim is reporting on... she is in the audience..

she worked out the ATPase chemomechanical system afffected by specific dictyostelium myosin II mutations...

jim recruited her  back to stfd  to  do this work...  she's a pedi cardiologist... heading up this program now..
lives on campus..

****

calcium comes in and get   motion of myosin on actin... one stroke...

the "burning of the ATP" fuel

the heads are where all the action is... do not need the rest... the motor IS the head domain..

all human cardiac myosin... can look at the effects of mutations to see what happens...

sl:  we have mad plasmids and have expressed the human beta-cardiac
myosin motor domain s1 carrying 5 HCM mutations and 5 DCM mutations.

•	Leslie Leinwand is the heavy wt on this work..

myosin, troponin etc  al give ries to HCM or DCM mutations..

tropomyosin  blocks the binding site on actin...

calcium  causes it to move  and expose the binding site..

Ruth Sommese is the lead on this work...

must look at actin/tropomyosin complex  together   (steric relations r important)

regulated thin filament  ATPase

correelaiton btwn  conc of Ca and  %ATPase activity...

HCM <=  hypercontractility of cells  (maybe)
DCM <=  hypocontrac  (maybe)  preliminary hypothesis.

you cannpt move faster than the time it takes to let go of the actin filament..

velocity of mvmt = 10 nano/ release time =  5 nano/
its just  distance = rate * time..

2 ways to change the force of contraciton..
1)  how many heads are generating force... how many are bound
eg abt  20percnt of heads are generating force (r bound)
2)  increase the strength of the force of each molecule...
eg mutations might be affecting the spring constant of a molecule;

use the laser trap  to actually measure the force gener. by a single molecule..

Jongmin Sung and Suman Nag... data collected at 3 AM today !!!!

laser trap single molec force measurement...
can mutate to incr force by 53%  !!!!

Mary Elting...  zero-mode  waveguide  to see indiv  atp ? binding..

what happends when  u put single molec in the context of a sarcomere...

IonOptix...  single cell trap...

mouse  cardiomyocytes...

we can demo Frank-Starling effect at the single cardiomyoscte level  showing stretch incr   force...

next, want to take induced pluripotent stem cell  iPS cells...

he ahs a 2nd lab in Bangalore in India...

therapeutic hypothesis

can one think abt therpaies yet???  there ar eno therapies so far for HCM/DCM...

normal oper range  pCA  goes from  10**-7  from 10**-6  see linear correl with ATPase activity  going from  0 to 100%

in HCM  the curve is shifted to the left...  that is  HCM is overly sensitive to pCa...  want to shift curve back toward the  right

therap hypthothesis

HCM mutations typic result in hypercontractility 2nd to incr sensitivaitonof the cardiac sarcomere to ca.

there are no known ca de-sensitizing drugs..

small molecl inhibition of the sarcomere activity will normlize ca sensitizationin HCM and ameliorate  the consequences of the HCM mutations.

******

Q (from introducer) how  wud  incr sensitivity lead to HCM?  or the reverse for DCM?  (HCM = hypertrophic cardiomyopathy)
DCM = dilated cardiomyop.

A  its preliminary...   even at cell level  u get  disrupted sarcomeres...  what cuases the disruption...
perhaps  actin filaments are breaking as a result of hypercontractile myosin... and hence get disrupted actin...

Q  Dan asks Q:  any mutations  assoc with change (diff) in stroke length...

A  in case of muscle myosin... stroke length is built into the molecule... depdns on
change btwn  bound with ATP vs unbound state...

comment from Kahty Ruppel...  does change in mice...

Q abt genetics of HCM...  simplet is autosomal dominant  but  lots of modif a nd lots of complexity.

Q/comment:  about TORQUE  ?  twisting moment  is sl different than  force...
invites Alex Dunn to comment..torque is due ot v small changes in bond length..

Q from Hawaiii... exac in puberty  etc...  how willit contrib to the unfolding of the phenotype
A  very complex... just working it out...  no hope of understanding it unless u understnd the single molec then single cell level...

assoc with CDI  is great (at stfd)

Q  environmental factors...  dilated cardiomyopathy...
viral infecitons  cause these...
infection, radiation
A  not on our radar yet

introducer = John Cook MD  cardiology... research in perph vas disease...