Abstract: The "growth cone" is the pathfinding organ of the neuron. It is the motile tip of the neuronal axon. It extends cellular processes, filopodia (dynamic cellular extensions containing actin bundles), that are essential for the axon to navigate to its proper destination. Little is known about the dynamics or signaling mechanisms, although a first step in initiating signal cascades is often filopodial adhesion. In contrast to the general assumption that all cell-substrate adhesions play equivalent roles, our studies establish that adhesions made by individual filopodia can mediate different and distinctive functions. The roles of filopodia and their adhesions in motility and guidance will be reviewed in this talk.

Adhesions at three sites in individual filopodia were found to have dissimilar functions. Tip adhesions suffice to signal. Adhesions made by single filopodial tips can initiate signal cascades that systematically alter cytoskeletal dynamics. Alterations are discrete, robust, and suffice to mediate specific growth cone turning behaviors. Basal adhesions form at nascent filopodial bases before filopodia emerge, remain at bases throughout filopodial lifetimes, and function in filopodial emergence and dynamics. They specifically associate with "focal rings," newly described organelles that link actin bundles to the basal adhesion and thereby mediate substrate anchorage. Focal rings also develop in Schwann cells and other cell types. Shaft adhesions lie along filopodial shafts, lack focal rings, and control the extent of lamellar ("veil") advance. Shaft adhesions inhibit veil advance. Veils are unaffected by basal adhesions, but readily advance along filopodia until they encounter shaft adhesions, where they stop advancing. Most intriguing, navigational cues can guide by targeting shaft adhesions. Filopodial tip adhesion to an inhibitory cue induces shaft adhesions and abolishes veil advance, whereas tip adhesion to a permissive cue prohibits shaft adhesions and promotes veil advance. Shaft adhesions can thus regulate both motility and navigation. The discovery of functionally distinctive adhesions compels a reevaluation of signaling mechanisms that were previously inferred under the assumption that adhesions are mono-functional. The discovery also shows that guidance responses are much more discrete and invariant than previously supposed, and are thus good candidates for mathematical modeling. Support: NSF-0212326.

Recent, relevant papers:

Steketee M., K.W. Tosney. (1999). Contact with isolated sclerotome cells steers sensory growth cones by altering distinct elements of extension. J. Neurosci. 19: 3495-3506

Polinsky, M., K. Balazovich and K.W. Tosney (2000). Identification of an invariant response: Contact with Schwann cells induces veil extension in growth cones. J. Neurosci. 20: 1044-1055.

Steketee, M., K.J. Balazovich and K.W. Tosney (2001). Filopodial initiation and a novel filament-organizing center, the focal ring. Mol. Biol. Cell. 12: 2378-2395.

Abstract: To properly segregate replicated chromosomes during mitosis requires the formation of a mitotic spindle, which consists of microtubules that emanate from the spindle poles and connect to chromosome-associated kinetochores. Kinetochores track along microtubule plus ends as the microtubules self-assemble and disassemble via dynamic instability. Due to the stochastic nature of microtubule dynamic instability, the sister kinetochores can transiently move away from each other, each kinetochore tracking along a disassembling microtubule. In this case, tension will develop between the kinetochores in the chromatin that links them together. Prior work suggested that tension influences the switching behavior associated with dynamic instability. We found that a Monte Carlo simulation model for microtubule dynamic instability that includes tension-mediated microtubule switching was consistent with experimental observations of both wild-type and replication-deficient GFP-tagged yeast kinetochores during metaphase. This model also requires that a stable spatial gradient of microtubule catastrophe rate exists, with a higher probability of catastrophe (stochastic switching from self-assembly to disassembly) occurring at the spindle equator than at the poles. Together, these processes can account for the spatial organization of yeast kinetochore microtubules and the results suggest that tension in the kinetochore-DNA complex promotes the stabilization of microtubules and protects them from disassembly.

Abstract: LAD-1, the sole homologue of the L1 family of neuronal cell adhesion molecules (L1CAMs), is required for nervous system development as well as embryogenesis. Indeed, we show that mutations in lad-1 result in Unc coilers that are Egl and constipated, as well as 40% embryonic lethality. Further analysis reveals misplacement of neuronal cell bodies in the mutants.

LAD-1 contains an ankyrin binding motif, FIGQY, which allows LAD-1 to bind UNC-44 ankyrin and be linked to the spectrin-actin cytoskeleton. We show that LAD-1 is phosphorylated at the tyrosine residue of the FIGQY motif; this phosphorylation event is dependent on the egl-15 FGFR-activated Ras pathway. Phosphorylated LAD-1 is localized to axon-muscle and epithelial adherens junctions that are free of non-phosphorylated LAD-1, suggesting distinct functions for phosphorylated LAD-1. Indeed, phosphorylated L1CAMs have been reported to bind doublecortin, a microtubule-associated protein. This suggests that phosphorylation is a mechanism for LAD-1 to switch from actin to microtubule cytoskeletal linkage.

Doublecortin is thought to play a role in neuronal migration. Thus, the biochemical interaction between doublecortin and L1CAMs is particularly intriguing in light of the neuronal misplacement defects observed in the lad-1 mutant. C. elegans contains a single doublecortin homologue, zyg-8, which was previously shown to play a role in mitotic spindle positioning. We show that the zyg-8 postembryonic mutants exhibit a similar phenotype to those of the lad-1 mutant: Unc and constipated coilers as well as high levels of embryonic lethality. This result suggests that the biochemical interaction between phosphorylated L1CAMs and doublecortin is functionally significant. We are in the process of genetically assaying if zyg-8 and lad-1 functionally interact.

Recent relevant publication:

Chen, L., Ong, B., and Bennett, V. 2001. LAD-1, the C. elegans L1CAM family homologue, has essential cell adhesion roles in the early embryo, participates in cell migration, and is a substrate for phosphotyrosine-based signaling. Journal of Cell Biology 154: 841-855.

Certain disorders in sexual development and reproductive function are traced to disorders in the rhythmic, pulsatile secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus. These disorders may require long-term hormone replacement therapy, and rhythmic delivery of GnRH is essential. Since GnRH is exceptionally potent, implantable hormone delivery systems may be considered. We are developing such a system, in which autonomous modulation of permeability of a hydrogel membrane to GnRH is driven by endogenous glucose, via a chemomechanical limit cycle established by feedback between the membrane and an enzyme. Several mathematical models of this system have been developed, with different levels of complexity. We will present results of a lumped, ODE-based model, for which the bifurcation structure has been worked out, and will also progress towards a more detailed, distributed (PDE-based) model.

We have attempted to develop a tissue-engineered artery and heart valve based on the approach of entrapping tissue cells within a forming collagen gel. The ability to harness the cell traction-induced contraction of the network of collagen fibrils to obtain the desired alignment of fibrils and cells will be described and explained. Recent efforts to drive "compositional remodeling" following the "structural remodeling" obtained via mechanically-constrained contraction, using fibrin as an alternative biopolymer to collagen for cell entrapment, with the goal of attaining the requisite mechanical properties, will be presented. Unlike the early "structural remodeling", the subsequent "compositional remodeling" and associated tissue growth that occurs in fibrin presents major modeling challenges.

Recent relevant publications:

A novel implantable collagen gel assay for fibroblast traction and proliferation during wound healing.
Enever PA, Shreiber DI, Tranquillo RT.
J Surg Res. 2002 Jun 15;105(2):160-72.

Short Course Slides/Materials click here
Group Photos