Growth cone motility.

Neurons obtain their stereotyped morphologies and connections as a result of growth cone migration. In the past year, studies on growth cone migration and pathfinding have helped to define certain properties of cytoskeletal filaments and cell membranes that may be important in growth cone function. Antisense mRNAs have proved to be particularly useful for examining the roles of specific neurite proteins.

Reactivation of newt lung cilia: evidence for a possible temperature- and MgATP-induced activation mechanism.

Optimal conditions have been developed for the isolation and reactivation of highly coupled, demembranated ciliary axonemes from newt lungs [Hard, Cypher, and Schabtach, 1988, Cell Motil. Cytoskeleton 10:271-284]. In the present study, the motility of these cilia was further characterized by examining the effects of nucleotides, divalent cations, and temperature on beat frequency. When exposed to a reactivating solution containing Mg2+ and ATP, nearly 100% of the axonemes were motile and beat at frequencies of 0-50 Hz, depending on [MgATP] and temperature. Divalent cations were required for movement, with Mg2+ 2-3 times more effective than Ca2+. There was no absolute requirement for Ca2+ for motility. The beat frequencies obtained with fixed ATP and varying Mg2+ concentrations indicate that MgATP serves as the actual substrate. The effects of MgATP on beat frequency depended on the degree of mechanochemical coupling and temperature. When highly coupled preparations were reactivated at 21 degrees C, double reciprocal plots of beat frequency vs. [MgATP] were biphasic with extrapolated Fmax values of 22 and 44.8 Hz. However, when reactivated at 10 degrees C and 30 degrees C, linear plots were generated with Fmax values of 18.3 and 48.9 Hz, respectively. The beat frequencies of cultured cells and reactivated axonemes also varied biphasically with temperature. Our data suggest that newt lung respiratory cilia possess an intra-axonemal activation mechanism involving a temperature- and MgATP-induced transition between two distinct states whose maximum beat frequencies differ by 200-300%.

Identification of cytoskeletal, focal adhesion, and cell adhesion proteins in growth cone particles isolated from developing chick brain.

Growth cones are intimately involved in determining the direction and extent of neurite elongation during development. They are able to monitor their environment and respond to it by undergoing directed motility. We have isolated a fraction enriched in growth cone particles from embryonic chick brain. Assayed by immunoblots, this fraction is enriched in GAP-43, and contains the cytoskeletal proteins actin, myosin II, neurofilament protein, tubulin, kinesin, and dynamin. All of the major components of focal adhesions are also present: alpha-actinin, vinculin, talin, and integrin. In addition to integrin, we also identify the cell adhesion molecules A-CAM, L1, fibronectin, and laminin in these particles. This preparation of isolated growth cone particles may be a useful model system for studying growth cone adhesion and motility.

Nerve growth cone motility.

Although many issues remain unresolved, the past year has witnessed a number of advances in our understanding of the inter-relationships between extracellular influences, cell phenotype, growth associated proteins, second messengers, and cytoskeletal components in the control of neurite outgrowth and growth cone behavior. Some of the early events associated with process initiation have been tentatively identified, and more is known about the assembly and stabilization of the microtubular framework of growing neurites. The mechanical forces involved in neurite extension have begun to be quantified, and interactions between the actin and microtubule systems are being further characterized. The current data more strongly support a functional role for GAP-43 in control of motility. The data also tend to support a central role for cytoplasmic calcium in mediating the actions of many growth-regulating influences, and strongly implicate changes in actin filament stability as mediating the behavioral effects of calcium.

Isolation and reactivation of highly-coupled newt lung cilia.

The paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5-5.0 cm in length. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12-13 micron in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X-100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21 degrees C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.

Taxol-induced anaphase reversal: evidence that elongating microtubules can exert a pushing force in living cells.

The effects of taxol on mitosis in Haemanthus endosperm were studied. Immuno-gold staining was used to visualize microtubules; observations on microtubule arrangements were correlated with studies in vivo. Mitosis is slowed down, but not arrested, by taxol over a wide range of concentrations. Taxol promotes the formation of abundant new microtubules and lateral association within and between microtubule arrays (spindle fibers). This leads to a pronounced reorganization of the spindle, especially at the polar regions. Chromosome arms may be pushed toward the equator in metaphase. Anaphase chromosomes, with their kinetochores still pointing to the poles, move backward before resuming their poleward migration. During anaphase, the interzone is depleted of microtubules and trailing chromosome arms are stretched and often torn apart by rapidly elongating polar microtubules. Fragments are transported away from the poles, apparently "riding" on the tips of microtubules. This provides evidence of "pushing" by elongating microtubules. The desynchronization of anaphase, often observed as one of the first effects of taxol, indicates that the anchorage of different kinetochore fibers varies. The data draw attention to modifications of spindle structure due to increased microtubule lateral associations and to the role of this process in spindle integrity and chromosome movement.