Zebrafish calsyntenins mediate homophilic adhesion through their amino-terminal cadherin repeats.

The calsyntenins are atypical members of the cadherin superfamily that have been implicated in learning in Caenorhabditis elegans and memory formation in humans. As members of the cadherin superfamily, they could mediate cell-cell adhesion, although their adhesive properties have not been investigated. As an initial step in characterizing the calsyntenins, we have cloned clstn1, clstn2 and clstn3 from the zebrafish and determined their expression in the developing zebrafish nervous system. The three genes each have broad, yet distinct, expression patterns in the zebrafish brain. Each of the ectodomains mediates homophilic interactions through two, amino-terminal cadherin repeats. In bead sorting assays, the calsyntenin ectodomains do not exhibit homophilic preferences. These data support the idea that calsyntenins could either act as adhesion molecules or as diffusible, homophilic or heterophilic ligands in the vertebrate nervous system.

The clustered protocadherins Pcdhα and Pcdhγ form a heteromeric complex in zebrafish.

The clustered protocadherin genes encode a diverse collection of neuronal cell surface receptors. These genes have been proposed to play roles in axon targeting, synaptic development and neuronal survival, although their specific cellular roles remain poorly defined. In zebrafish there are four clustered protocadherin genes, two pcdhα clusters and two pcdhγ clusters, that give rise to over 100 distinct proteins, each with a distinct ectodomain (EC). The zebrafish is an excellent model in which to address the function of protocadherins during neural development, as the embryos are transparent, develop rapidly, and are amenable to experimental manipulation. As a first step to investigating the clustered protocadherins during zebrafish development, we have generated antibodies against the common cytodomains of zebrafish Pcdhγ. We compare the distribution of Pcdhγ with Pcdhα and find a similar pan-neuronal pattern, with strong labeling of neurons within all major regions of the central nervous system. Pcdhα and Pcdhγ are particularly enriched in the developing visual system, with strong labeling found in the synaptic layers of the retina, as well as the optic tectum. Consistent with studies in mouse, we find that Pcdhα and Pcdhγ are present in a complex, as they can be co-immunoprecipitated from zebrafish larval extracts. This interaction is direct and occurs through the ECs of these proteins. Using standard bead aggregation assays, we find no evidence for intrinsic adhesive ability by either Pcdhγ or Pcdhα, suggesting that they do not function as cell adhesion molecules.

Differential expression, alternative splicing, and adhesive properties of the zebrafish δ1-protocadherins.

Protocadherins comprise the largest family within the cadherin superfamily of cell surface receptors. Here, we characterize the δ1-protocadherin subfamily during the development of the zebrafish nervous system. In zebrafish, there are five δ1-protocadherins: pcdh1a, pcdh1b, pcdh7a, pcdh7b, andpcdh9. Each protocadherin gene is highly homologous to its human ortholog. While the expression pattern in the developing CNS is similar for each δ1-protocadherin, with labeling observed in all major subdivisions, the detailed patterns are distinct. In addition, we provide evidence for alternative splicing of the pcdh7b and pcdh9 genes, resulting in variation in their respective cytoplasmic domains. As protocadherins are widely regarded to act as cell adhesion molecules, we used in vitro assays of δ1-pcdh ectodomains to directly test their adhesive properties. We found no evidence for calcium-dependent, homophilic adhesion, contrasting sharply with the behavior of classical cadherins.

Growth cone and dendrite dynamics in zebrafish embryos: early events in synaptogenesis imaged in vivo.

We used time-lapse fluorescence microscopy to observe the growth of Mauthner cell axons and their postsynaptic targets, the primary motor neurons, in spinal cords of developing zebrafish embryos. Upon reaching successive motor neurons, the Mauthner growth cone paused briefly before continuing along its path. Varicosities formed at regular intervals and were preferentially associated with the target regions of the primary motor neurons. In addition, the postsynaptic motor neurons showed highly dynamic filopodia, which transiently interacted with both the growth cone and the axon. Both Mauthner cell and motor neurons were highly active, each showing motility sufficient to initiate synaptogenesis.

The motor protein myosin-I produces its working stroke in two steps.

Many types of cellular motility, including muscle contraction, are driven by the cyclical interaction of the motor protein myosin with actin filaments, coupled to the breakdown of ATP. It is thought that myosin binds to actin and then produces force and movement as it 'tilts' or 'rocks' into one or more subsequent, stable conformations. Here we use an optical-tweezers transducer to measure the mechanical transitions made by a single myosin head while it is attached to actin. We find that two members of the myosin-I family, rat liver myosin-I of relative molecular mass 130,000 (M(r) 130K) and chick intestinal brush-border myosin-I, produce movement in two distinct steps. The initial movement (of roughly 6 nanometres) is produced within 10 milliseconds of actomyosin binding, and the second step (of roughly 5.5 nanometres) occurs after a variable time delay. The duration of the period following the second step is also variable and depends on the concentration of ATP. At the highest time resolution possible (about 1 millisecond), we cannot detect this second step when studying the single-headed subfragment-1 of fast skeletal muscle myosin II. The slower kinetics of myosin-I have allowed us to observe the separate mechanical states that contribute to its working stroke.

Three-dimensional structure of Acanthamoeba castellanii myosin-IB (MIB) determined by cryoelectron microscopy of decorated actin filaments.

The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a "classical" myosin-I, Acanthamoeba myosin-IB (MIB), at approximately 18 A resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, approximately 10 degrees, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.

Brush border myosin-I structure and ADP-dependent conformational changes revealed by cryoelectron microscopy and image analysis.

Brush border myosin-I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751-753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain-binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The approximately 31 degrees swing corresponds to approximately 63 A at the end of the rigid light chain-binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.

Three-dimensional structure of Brush Border Myosin-I at approximately 20 A resolution by electron microscopy and image analysis.

Brush Border Myosin-I (BBMI) is a single-headed, unconventional myosin found in the microvilli of intestinal epithelial cells where it forms lateral bridges between the core bundle of actin filaments and the plasma membrane of the microvillus. A three-dimensional (3D) reconstruction of BBMI was made from images of negatively stained, two-dimensional (2D) crystals grown on lipid monolayers formed from mixtures of phosphatidylserine and phosphatidylcholine. The resolution of the 3D map extends to approximately 20 A and allows identification of all of the major structural domains of BBMI. The BBMI molecule is composed of three domains: a globular motor domain, a light-chain-binding domain and a lipid-binding domain. In our map, the putative motor domain is connected to an extended density, which we believe to be the light-chain-binding domain. This long, narrow region has three distinct bends, which may delineate the bound calmodulin light chains. Following the last calmodulin there is density which extends for a short distance across the lipid surface and is presumably the carboxy-terminal lipid-binding domain.

Kinetic characterization of brush border myosin-I ATPase.

Brush border myosin-I (BBM-I) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells. We used stopped-flow kinetic analysis to measure the rate and equilibrium constants for several steps in the BBM-I ATPase cycle. We determined the rates for ATP binding to BBM-I and brush border actomyosin-I (actoBBM-I), the rate of actoBBM-I dissociation by ATP, and the rates for the steps in ADP dissociation from actoBBM-I. The rate and equilibrium constants for several of the steps in the actoBBM-I ATPase are significantly different from those of other members of the myosin superfamily. Most notably, dissociation of the actoBBM-I complex by ATP and release of ADP from actoBBM-I are both very slow. The slow rates of these steps may play a role in lengthening the time spent in force-generating states and in limiting the maximal rate of BBM-I motility. In addition, release of ADP from the actoBBM-I complex occurs in at least two steps. This study provides evidence for a member of the myosin superfamily with markedly divergent kinetic behavior.

Two-dimensional crystallization of brush border myosin I.

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22121 or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 A and show that the asymmetric unit is the same in both crystal types.

A 32 degree tail swing in brush border myosin I on ADP release.

Brush border myosin I (BBMI) is a single-headed, unconventional myosin from intestinal microvilli, composed of a heavy chain of relative molecular mass 119,000 (M(r) 119K) and three calmodulin light chains. Although believed to have a largely structural role, it exhibits the normal actin-activated ATPase and motility properties of a member of the myosin superfamily. Here we present three-dimensional maps of BBMI-decorated actin filaments with and without bound MgADP. While the motor domain remains in a state similar to rigor, the light-chain-binding domain swings through approximately 32 degrees, resulting in a approximately 50-A movement at the end of the region visualized (the second calmodulin light chain). This could correspond to approximately 72-A movement of the entire domain. Although qualitatively similar to the movement observed in myosin II, the magnitude of the change is sufficiently different to suggest that structural changes during the actomyosin ATPase cycle differ among myosins, possibly reflecting adaptation for specialized functional demands.

Theories of muscle contraction.

A survey of the mainstream theories in the modern study of the mechanism of muscle contraction is made, with particular emphasis placed on the experimental results which most influenced the progression of ideas. Starting with early elastic and viscoelastic theories of muscle contraction, a chronological organization is used to present, in detail, the results leading up to the swinging crossbridge model. A brief review is made of the experimental results modifying the original crossbridge model such as transient-state mechanics, in vitro kinetics, and kinetic measurements performed on demembranated muscle fibers. Following a brief synopsis of three of the more prevalent alternative models, a summary of the more relevant structural studies is presented. Finally, recent results pertaining to the mechanism of muscle contraction are presented and their promise for the future is discussed.