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Figure 1. Basic functions for plus and minus end-directed myosins in cells. Myosin drives oriented transportation of cargo (A) and sliding of actin filaments (B). Myosin head (black balls) and tail (black stick) domains, actin filaments (red, blue lines), vesicle (circle), plasma membrane (curved and straight lines). Both indicated types of actin organization (A and B) exist in nonmuscle motile cells. A, Myosin pulls cargo on a uniform filament polarity actin network. Plus end- (upward arrow) and minus end- (downward arrows) directed myosins transport cargo in opposite directions. Myosins I and V are known plus end-directed transport motors. Myosin VI, the minus end-directed actin motor, also has transport motor activity in cells, but oriented movement of cargo remains to be demonstrated (see text). B, Myosin slides actin filaments in an opposite filament polarity (antiparallel) actin network. A plus end-directed myosin (top) pulls actin filaments together (thick arrows move together), generating pulling or contraction force in the cell. A minus end-directed myosin (bottom) pushes filaments apart (thick arrows move apart), generating pushing or expansion force in the cell. Contraction force generated by myosin II has been documented in many systems. However, expansion force resulting from minus end-directed myosins is, at this point, hypothetical.
- Two distinct types of actin organization that exist in cells, uniform and opposite filament polarity actin networks (see below for descriptions), allow two extreme types of myosin function, respectively (Fig 1): oriented transportation of cargo (Fig 1 A) and sliding of actin filaments (Fig 1 B).
- To drive cargo transport, myosin (Fig 1 A, single black ball and stick) moves (Fig 1 A, arrows) attached cargo (Fig 1 A, circles, curved line) over the surface of a uniform filament polarity actin network (Fig 1 A, red lines).
- In this actin organization, all actin filament plus ends face towards the cell surface (Fig 1 A, as indicated).
- This allows oriented transportation of cargo into or out of the cell (e.g., Fig 1 A, compare upward and downward arrows).
- Thus, a plus end-directed myosin will transport attached cargo outwards towards the cell surface, such as a vesicle on the exocytic pathway (Fig 1 A, upward arrow).
- Conversely, a minus end-directed myosin will transport attached cargo inwards, away from the cell surface, such as a vesicle on the endocytic pathway or a region of the plasma membrane that is being retracted or tethered by the myosin (Fig 1 A, downward arrows).
- To drive actin filament sliding, myosin oligomers (Fig 1 B, two black balls and stick) sit between repeating units of actin filament subbundles (e.g., two subbundles are represented by red and blue lines in Fig 1 B) in an opposite filament polarity actin network, and move subbundles relative to one another (e.g., Fig 1 B top, compare thick arrows).
- In this actin organization, actin filament plus ends from adjacent subbundles face opposite directions (Fig 1 B, compare red and blue lines) and myosin sits between overlapping/interdigitating filament minus ends from adjacent subbundles (Fig 1 B, as indicated).
- Also, net filament plus ends are anchored to the plasma membrane (e.g., Fig 1 B, junction of vertical and horizontal lines) via adhesion complexes or other connections.
- Thus, filament sliding driven by a plus end-directed myosin in an opposite filament polarity actin network exerts net pulling force or cell contraction force (Fig 1 B, upper panel, arrows move together).
- Conversely a minus end-directed myosin in the same actin network exerts net pushing force or cell expansion force (Fig 1 B, bottom, arrows move apart).
The Journal of cell biology.
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Figure 2. Biological processes requiring myosin VI activity. Simplified views of pseudocleavage (A–C, only two nuclei are shown), sperm individualization (D–F, only 3 out of 64 sperm are shown) in direction of arrowheads (E), and stereocilia development (G–I) representing early (A, D, and G) and late (B, E, and H) stages, and defects (C, F, and I) in these processes when myosin VI activity is blocked. Black lines (plasma membrane); black ovals and wavy lines (nuclei); red lines (actin filaments); blue lines (microtubules); green circles (localization of myosin VI); particle motility (A, arrows) before pseudocleavage, cytoplasmic bridge (e.g., asterisks in E), and accumulation of waste (E, hash) in sperm. Note when myosin VI activity is blocked, the position of plasma membrane in sperm (F) and organization of actin in stereocilia (I) has not been described and is theoretical as shown.
- Three actin-based shape change events known to require myosin VI activity (Fig 2) are: pseudocleavage in fly embryo blastoderm (Fig 2, A–C); sperm individualization in flies (Fig 2 D-F); and development of stereocilia in mouse hair cells (Fig 2, G–I).
- Whereas these shape change events differ in scale and gross morphology, a common feature is invagination of the plasma membrane (Fig 2, compare B, E, and H).
- When myosin VI activity is blocked in these processes, related defects in membrane invagination and other events occur (Fig 2, compare C, F, and I) and argues that myosin VI function in these tissue types is similar in at least some respects.
- Pseudocleavage describes invagination of plasma membrane to keep mitotic spindles in the syncitium separate during mitosis (compare Fig 2A and Fig B).
- When myosin VI activity is blocked with antibodies, membrane invagination (the pseudocleavage furrow) is less deep and mitotic spindles fuse (Fig 2 C; Mermall and Miller 1995 ).
- Myosin VI generally transports particles in the actin cortex of syncitial blastoderm (Fig 2 A, arrows; Mermall et al.
- Before mitosis, myosin VI–containing particles are relatively dispersed (Fig 2 A, green circles).
- At mitosis, these particles relocate and concentrate in pseudocleavage furrows (Fig 2 B, green circles).
- It is unlikely that myosin VI functions to directly transport particles that might contain important molecules to pseudocleavage furrows at mitosis: there are no oriented actin tracks leading to furrows and, moreover, observed myosin-VI driven particle motility is nonoriented (Fig 2 A, compare arrows).
- In Drosophila testis, the process of individualization simultaneously converts a network of interconnected sperm in a syncitium into individual sperm cells (Fig 2, compare D and E).
- Individualization is driven by an actin structure, termed the investment cone (Fig 2 E, red lines), which travels from the head to the tail of each sperm (Fig 2 E, in direction of arrowheads).
- As the cone progresses, plasma membrane invaginates at the sperm connection sites (cytoplasmic bridges, two are denoted with asterisks in Fig 2 E) so that each sperm is encased in its own membrane (Fig 2 E, compare above and below the investment cone).
- 1999 ; Fig 2 F, compare red lines and black ovals).
- Stereocilia are finger-like structures that transduce sensory information on the apical surface of hair cells (Fig 2 H).
- Each stereocilium comprises a bundle of actin filaments (Fig 2 H, red lines) that extend below the apical surface to form actin rootlet anchors (Fig 2 H, splayed red lines).
- In mice effectively null for myosin VI, stereocilia are normal before birth (Fig 2 H).
- Starting at birth in mutants (Fig 2 I), the apical plasma membrane (black line) looses its position and rises up between adjacent stereocilia.
- 1999 ; Fig 2, compare H and I).
- By about two weeks after birth, fused giant stereocilia structures have formed in mutant mice (Fig 2, compare H and I; Self et al.
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Figure 3. Transport motor functions for myosin VI during sperm individualization. Myosin VI transports plasma membrane (A) or recycling membrane vesicles (B) during individualization. Both models account for observed accumulation of myosin VI towards the bottom of actin investment cones, and use syncitial plasma membrane as a required supply of membrane (total surface area is {~}1.2–2.4 x 105 µm2 around 64 sperm in the syncitium, and 1.4 x 105 µm2 for 64 individualized sperm.; calculated from data presented in Tokuyasu et al. 1972 ), which is consistent with observed shrinkage of sperm circumference after individualization. Left, Top view of sperm; right, side view of sperm; asterisk, feduciary mark on cytoplasmic bridge; blue lines and circles, microtubule axoneme; long thick arrows, direction of investment cone motility and individualization. A, Myosin VI (green ball and stick) tethers syncitial plasma membrane (black lines) onto investment cones (red lines). As myosin VI (short arrows) and/or the investment cone move downwards, attached syncitial plasma membrane is consequently pulled downwards (arrowheads), thus resulting in membrane invagination. B, Endocytosis (black circles) removes plasma membrane from the syncitium. Myosin VI (green ball and stick) transports endocytic membrane vesicles across the investment cone (red lines). Exocytosis (long thin arrows) and membrane invagination (dashed line) occurs subsequently. In a related mechanism, myosin VI is instead placed on the exocytic pathway (Hicks et al. 1999 ).
- Myosin VI may function to transport membrane in one of two distinct pathways (Fig 3, compare A and B) that use syncitial plasma membrane as a supply for required membrane invagination at cytoplasmic bridges.
- In the plasma membrane transport model (Fig 3 A), myosin VI (Fig 3 A, green balls) binds syncitial plasma membrane (Fig 3 A, black lines) and directly participates in membrane invagination by repositioning (tethering) this membrane towards the bottom of actin investment cones (Fig 3 A, in direction of short arrows).
- In the membrane vesicle transport model (Fig 3 B), myosin VI (Fig 3 B, green balls) instead binds endocytic vesicles (Fig 3 B, circles) derived from syncitial plasma membrane and moves them (Fig 3 B, short arrows) across the investment cone (Fig 3 B, red lines).
- This provides required membrane at cytoplasmic bridges for subsequent exocytosis and membrane invagination (Fig 3 B, dashed line).
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Figure 4. Two distinct functions for Myosin VI in stereocilia organization. Myosin VI functions as a motor to transport plasma membrane (A) and generate expansion force (B) in hair cells. Actin filament minus ends face towards rootlets (Tilney et al. 1980 ) and arguably interdigitate where rootlets splay (as indicated, thin black, red, and blue lines). A, Myosin VI (single green ball and stick) tethers (short arrows) the apical cell surface (thick black line) onto stereocilia rootlets to maintain surface membrane topology. B, Myosin VI (two green balls and stick) slides interdigitating filament–minus ends apart between adjacent stereocilia rootlets (long arrows move apart). Opposing pushing forces are exerted on each rootlet (e.g., long arrows acting on the middle rootlet), which keeps each stereocilium in place. Net outwards force is exerted on peripheral rootlets due to fewer nearest neighbors (e.g., long arrow acting on the left and right rootlet, respectively) causing stereocilia to tilt inwards above the cell surface.
- In this membrane transport model (Fig 4 A), myosin VI (Fig 4, single green balls) binds apical plasma membrane (Fig 4, thick black line) and moves attached membrane downwards a short distance (Fig 4, short arrows) towards actin filament minus ends in rootlets, thereby effectively tethering the plasma membrane at the base of stereocilia.
- Alternatively, myosin VI generates expansion (pushing) force between adjacent stereocilia rootlets (Fig 4 B) to directly stabilize stereocilia.
- Because each stereocilium is surrounded by several neighbors, opposing pushing forces are exerted on each stereocilium rootlet, thus keeping each stereocilium in place (e.g., Fig 4 B, opposing long arrows acting on the middle stereocilium).
- That this anchoring may come from rootlet-pushing, but not pulling, explains why mature stereocilia tilt towards each other above the cell surface (e.g., Fig 4).