Cell Motility and The Cytoskeleton

Biology 574; Fall 2000                Prof. Peter K. Hepler and Guests

Unconventional Myosins

Myosin-V

• The most extensively studied of the unconventional myosins
• Most organisms have at least one member
• Myosin-V is a fundamental component of organelle transport
• Possible cargoes are melanosomes, synaptic vesicles, vacuoles and mRNA
• Differs structurally from other myosins
• Extended neck domain
• Tail that allows dimerization
• Does not form filaments due to a globular carboxy-terminus

Model based on rotary-shadowed electron micrographs and primary structure analysis

• Myosin V diverged from other members before the divergence of yeast and animals
• There are orthologues and paralogues

• The mutation in coat color dilute was propagated by mouse fanciers at the turn of the 19th century
• The chromosomal alteration was the result of a ectopic insertion of a provirus that allow the cloning of the gene
• The second member was cloned from rats by degenerate PCR, each has 77% similarity in the head domain and 78% overall
• Myo5a and Myo5b in mammals

• Temperature sensitive cell cycle mutant in S. cerevisiae, unbudded morphology at restrictive temperature
• Complementation showed a myosin-V (called myo2p, second myosin from yeast)
• Another myosin-V was identified by probing cDNA libraries, (myo4p)
• The tails are shorter and there are fewer regions of coiled coil

• Identified biochemically in chicken brains, called p190
• After cloning it was showed:
• Yeast, chicken and mammals myosin-V constitute a separate class of myosins
• Imperfect tandem repeats present in all the necks, coined the term IQ motif
• Calmodulin can bind to IQ motifs

• In squid myosin-V was identified in axoplasm
• The protein purified and partially sequenced
• Associates with endoplasmic reticulum
• Antibodies against it inhibit vesicle transport
• In Drosophila the third IQ motif starts with LS, unique to Drosophila
• There could be specific light chains
• C. elegans’ myosin-V is as divergent as the other members
• Plant myosins are closely related to myosin-V, they have been assigned their own classes which are: myosin VIII, XI and XIII

 

Head

• Force generated by the head
• The ATPase activities and actin-binding affinities vary widely in other myosins
• All characteristics of myosin-V are based on chicken myosin
• Some properties are apparently unique to myosin-V
• The ATPase is described by its K⁺-EDTA-ATPase and Mg²⁺-ATPase
• Influence by F-actin and Ca²⁺ as modulators
• Ca²⁺ (between 1-3 mM) plus F-actin stimulates the ATPase
• Ca²⁺ influences the rate of movement
• Increase of Ca²⁺ to 10 mM decreases velocity immediately followed by a gradual decay
• Spiking with calmodulin prevents the gradual decay but not the immediate decrease
• Barbed end directed motor at 450-600 nm/sec
• Seems that Ca²⁺ shifts myosin-V to an extremely low gear, which could provide greater force to transport large cargoes

Neck

• An a-helix with sequences that bind light chains
• In conventional myosins, the light chains contribute to the rigidity and stability of the helix
• The known light chains in myosin-V are members of the EF hand family
• Quick freeze, deep-etch electron micrographs show a thickened segment
• The peptide sequence is an imperfect repeat of ‘IQxxxxRGxxxR’ or IQ motif
• Myosin V has 6 of these repeats, the most carboxy terminal being the most divergent
• Evidence that calmodulin binds to the IQ motif:
• Initially calmodulin was copurified at a ratio of 4:1
• Immunofluorescence in yeast colocalized it with calmodulin
• In gel overlays,  overexpressed IQ motifs can bind calmodulin
• Immunoprecipitation of myo2p precipitates calmodulin with or without Ca²⁺
• Two other proteins copurify and are the essential light chains of myoisn II

Tail

• Probably where it interacts with its cargo
• The identity of the cargo would define where the myosin apply the force, hence its function
• Possible cargoes identified in neurons, pigment cells and yeast
• All mutations of Myo5a have dilution of coat color, most alleles have neurological phenotypes
• Transcript present in every organ but liver in adult mice
• Present in every neuron in the brain

Function in neurons

• Physiological rather than developmental role
• Probably associated with vesicles
• Punctate staining in neurons
• Found associated with synaptic vesicles by immunoprecipitation
• During purification of brain myosin-V, a brain vesicle fraction is generated
• In squid axoplasm immunoelectron microscopy showed myosin V and kinesin in the same vesicles
• Two-hybrid interaction between the tail of myosin V and kinesin

Function in melanosomes

• Pigment is synthesized in melanosomes by melanocytes and transported to the hair bulb via dendritic processes
• dilute melanocytes do not have dendrites, but myosin-Va  is required during melanosome transport
• Myosin-Va colocalizes with melanosomes and captures them in the periphery of the melanocyte
• Myosin-V has cell type-specific cargoes
• Transcripts of myosin-Va have tissue-specific splicing pattern

S. cerevisae

• Temperature sensitive myo2-66
• Accumulation of vesicles
• Abnormal F-actin structures
• Disruption of directed growth
• Delocalization of chitin
• Double mutants placed myo2p at the post-Golgi stage of secretion
• Cells do not transfer vacuoles to the daughters and colocalization with the vacuole
• A point mutation at the tail disrupts localization and vacuole inheritance
• Myo4p is involved in mating type switching
• Cleavage of DNA at the MAT locus by HO endonuclease restricted to the mother cell
• Daughter cells are protected by expressing Ash1p, a HO transcriptional repressor
• ASH1 mRNA accumulation in daughter cells is dependent on actin and Myo4p
• Myo4p does not affect vacuole inheritance and does not rescue loss of Myo2p
• Loss of Myo2p can be rescued by SMY1, a member of the kinesin superfamily
• Interesting since in animals there is cooperation between myosin-V and microtuble based motors

For the future

• Sequences that interact with phospholipids are not present in the tail
• It is likely that it interacts with cargo via an intermediate protein
• The protein AF-6 has a homologous domain to the tail of myosin-V plus a membrane localization domain (PDZ), this protein could compete for cargo and release it next to the membrane

Myosin-V is a processive motor

	Myosin-V head and neck at low [ATP] on F-actin visualized by EM Images from: Matthew L. Walker, Stan A. Burgess, James R.Sellers, Fei Wang, John A. Hammer III, John Trinick & Peter J. Knight. Two-headed Binding of a Processive Myosin to F-actin. Nature, 405, 804-807  (2000) http://www.leeds.ac.uk/bms/research/muscle/muscle.htm
	Pre-power stroke attachment The detached head sweeps over a wide angle, suggesting that the junction between the heads is flexible. Notice that the detached head cannot approach the next binding site while the attached head is in the pre-power stroke position. Frame width is about 65 nm.
	Post-power stroke attachment Notice the wide sweep of positions the detached head can adopt, and that it cannot get close to its former attachment site once the attached head has undergone the power stroke. Frame width is about 65 nm.

Molecular model of myosin-V from Vale and Milligan
 

• Unlike myosin II, myosin V is processive: one molecule can undergo multiple steps before it detaches from its track
• Feedback enhanced optical trap to examine stepping kinetics
• Analysis of the distribution of time periods separating 36nm steps
• Number and duration of rate-limiting transitions preceding each step
• Tightly coupled motor with a cycle time limited by ADP release
• Tight chemomechanical coupling
• ADP release is rate limiting
• ATP promotes fast dissociation
• Phosphate release occurs quickly after actin binding
• Hydrolysis step is fast
• ADP state has high actin affinity

A possible model for processivity

• Myosin V dwells with both heads attached, leading head in ADP, trailing head in rigor, ATP binding to trailing head promotes dissociation from actin (i)

• Forward movement of the released head discharges molecular strain (i,ii)

• The previous leading head becomes the trailing head, newly detached leading head quickly hydrolyzes ATP and binds actin

• Force generation follows either actin binding or phosphate release, which occurs with actin binding or immediately after (iii,iv)

• These steps are fast compared with ADP release

• At this point the molecule is in its kinetically dominant state: both heads bound to actin and ADP, leading one prestroke-like and trailing one in poststroke-like

• The leading head is stressed against the direction of motion, the trailing one along, this asymmetry could bias ADP release from the trailing head, but intramolecular strain does not affect the ADP step directly.

• Force is likely to slow another transition in the leading head, like isomerization between a state that releases ADP very slowly and a state competent to release ADP at 13 s^-1

• Stress provides an elegant way for distant heads to communicate in the large myosin-V

Myosin I

• Low molecular weight motors
• Expressed in a wide range of organisms: protozoa, fungi, nematodes and mammals
• Typically associated with membranes
• All members have a tail domain rich in basic residues to bind anionic phospholipids
• Important roles in moving membranes against actin and membrane-actin interactions

Properties

• First unconventional myosin to be discovered, by Tom Pollard in 1970s  (story by Tom Pollard)
• Conserved myosin motor domain at the N-terminus
• Neck region with 1-6 IQ motifs
• C-terminus tail rich in basic residues (polybasic domain)
• Four distinct subclasses, referred by the name of the best studied in the group

Subclass 1 (Ameboid myosin I)

• The tail is composed of three distinct domains
• 1st the polybasic, common to all myosin-Is, which mediates binding to membranes and anionic phospholipids in vitro
• 2nd rich in glycine, proline and alanine (or glutamate, or serine) called GPA domain, binds to actin in a ATP insensitive manner
• 3rd a src homology 3 (SH3) domain at the C-terminus, protein module that mediates protein-protein interaction in signaling pathways associated with the plasma membrane and the actin cytoskeleton
• Has one or two IQ motifs at the neck
• In protozoa has calmodulin related light chains, in animals probably calmodulin
• The actin-activated Mg²⁺-ATPase and the in vitro motility are regulated by phosphorylation of a serine or threonine in the motor domain, but higher eukaryotes have a negatively charged residue instead, and probably are regulated by Ca²⁺ calmodulin
• The Acanthamoeba and Dictyostelium heavy chains are phosphorylated by a kinase related to PAK that requires small G-proteins for activation
• Localizes to the plasma membrane and intracellular vesicles in a range of cell types
• Localizes to actin patches in yeast, and in the contractile vacuole of Acanthamoeba

Subclass 2 (Brush Border Myosin I)

• Initially identified in the microvilli of chicken intestinal epithelial cells, where are the lateral links between the plasma membrane and the core of actin filaments in the microvilli
• Expressed in a range of different tissues
• Single heavy chain with 3-6 IQ motifs, each binds one calmodulin, regulated in Ca²⁺ calmodulin dependent manner
• The C-terminal tail only a polybasic domain
•  Maximum Mg²⁺ at 1-2 mM Ca²⁺, decreases at 10 mM
• Also present in Golgi-derived vesicles, could play a role in the apical transport of membrane in epithelia

Subclass 3 (Myosin Ib)

• Originally purified from bovine adrenal medulla and cloned from rat brain, Drosophila and frog
• Resembles brush border myosin-I
• 3 IQ motifs, tail with a polybasic domain
• Ca²⁺ increases the activity of the actin-activated Mg²⁺-ATPase
• Largely membrane associated (ER and plasma membrane), but also soluble
• Expressed in a wide range of tissues

Subclass 4 (Myr4)

• Members found in rat, Drosophila and C. elegans
• 2 IQ motifs that bind calmodulin
• Tail region enrich in basic and hydrophobic residues
• Present in different tissues in rat
• Possibly associated with organelles
• In Drosophila only found in the developing and mature gut

Myosin I Mutants

• Null mutants in: Dictyostelium, Emericella nidulans and yeast
• Essential for cellular processes that require the functioning of the actin-rich cortex of the cell
• Deletion of 1 gene in E. nidulans or 2 genes in yeast is lethal
• Mutant fungi are defective in secretion and endocytosis
• Yeast mutants have altered actin distribution
• Dictyostelium mutants are impaired in uptake of nutrients by non-receptor mediated fluid phase pinocytosis, but viable
• Acanthamoeba cells loaded with antibodies that inhibit myosin IC show defects in osmoregulation

Myosin VI

• Little cell biological evidence of a pointed end directed motor
• Searching for a unusual insert adjacent to the motor domain

• Humans express ~30 different myosin genes, ~20 are unconventional of ~10 classes
• A typical non-muscle cell expresses one or two conventional myosins but more than 10 unconventional
• Model of myosin: small nucleotide-dependent changes in the head are transferred via a ‘converter’ domain to the neck, which acts as a rigid lever arm to amplify the change
• Only class-VI myosins have a larger insertion between the head and the neck
• Class-VI myosins first discovered in Drosophila, 95F for its chromosomal location, two classes in C. elegans and fish
• In mammals was first identified in pigs and it is also the defective gene of Snell’s waltzer mice which causes deafness and vestibular problems because of defects in the hair cells of the inner ear
• Abundant protein (0.15-0.8% of total protein), expressed in most cells and tissues
• Not identified in Dictyostelium and not present in yeast

• Head shares 35% identity with muscle myosin-II
• Ends with a 53 amino acids insertion
• The neck has 1 IQ repeat
• The tail has a coiled coil and a globular domain
• Probably a two headed dimer (~140 kDa each) with two calmodulin light chains

To show the backward motion

• Coexpression of myosin-VI head and neck with calmodulin (analogous to S1)
• Epitope tagg to link the myosin to the coverslip
• Use of actin filaments polymerized from rhodamine-actin nuclei as polarity markers
• The myosin-VI moved toward the pointed end
• Myosin-V moved in the opposite direction
• Also was the first evidence that myosin-VI can function as a motor
• Slow, sliding velocity of ~60 nm/sec

Explanations for backward movement

• Retain basic structure of the motor, and rearrange the hinge near the converter to reverse the swing, the 53 amino acids insert could do this
• Use of cryoelectron microscopy to visualize the position of myosin-VI either with ADP or in rigor (post-stroke, nucleotide free)
• The light chain domain is held at a different angle in rigor than other myosins and points toward the pointed end ~145 deg.
• Only a 15-20 deg. swing between the ADP and rigor, still necessary to see the pre-stroke state
• Also need of crystallographic studies of the insert

• Myosin-VI has a phosphorylatable residue at the TEDS-rule site in the head domain
• Myosin-I is activated by phosphorylation at this domain, myosin-VI could be too
• The calmodulin light chain could render it Ca²⁺ regulated
• Important to determine if is processive since it is believed to move organelles
• The short neck of myosin-VI suggests it will spiral along the actin cable

Localization and function

• Only abnormalities in Snell’s waltzer mice are defect in hearing and balance
• Myosin-VI is abundant in actin-rich regions of the hair cells, cuticular plate and pericuticular necklace
• Plays a role in maintaining the structural integrity of the individual stereocilia
• Might help to hold down membranes between the cilia, ideal for a pointed end motor since the barbed ends of the bundles point towards the membrane
• Myosin-VI could also transport the membrane components necessary to maintain the stereocilia
• Also found in the terminal web at the base of microvilli in the intestinal brush border

• In Drosophila 95F is produced in many tissues
• Important in syncytial blastoderm, associates with uncharacterized particles that translocate to the pseudocleavage furrows
• Important to transport cytoplasmic particles form nurse cells to the oocyte

• Membrane remodeling in the individualization of adjacent spermatids in the fly testes
• Mutation of one of the myosin-VI of C. elegans also causes defects in spermatogenesis
• Roles in membrane-cytoskeleton interactions and organelle or particle transport

Membrane remodeling in the individualization of adjacent spermatids in the fly testes

References

V. Mermall, P. L. Post and M. S. Mooseker (1998) Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science 279: 527-33

D. W. Provance and J. A. Mercer (1999) Myosin-V: head to tail. Cell. Mol. Life Sci. 56: 233-242

M. Rief, R. S. Rock, A. D. Mehta, M. S. Mooseker, R. E. Cheney and J. A. Spudich (2000) Myosin-V stepping kinetics: A molecular model for processivity. Proc. Natl. Acad. Sci. U. S. A. 97: 9482-9486

O. C. Rodriguez and R. E. Cheney (2000) A new direction for myosin. Trends Cell Biol. 10: 307-311

M. A. Titus (1999) Myosin I. in: Guidebook to the Cytokeletal and Motor Proteins. T. Kreis and R. Vale edit. 2nd ed. Oxford University Press Inc. New York pp. 430-434

R. D. Vale and R. A. Milligan (2000) The way things move: Looking under the hood of molecular motor proteins. Science 288: 88-95

M. L. Walker, S. A. Burgess, J. R. Sellers, F. Wang, J. A. Hammer, J. Trinick and P. J. Knight (2000) Two-headed binding of a processive myosin to F-actin. Nature 405: 804-807
 

View links to recent papers on myosin structure from the PUBMED database