Timothy M. Gomez

 

 

Regulation of Axon Guidance by Second Messengers

E-mail: tmgomez@wisc.edu

Research Strengths: Development: Plasticity and Repair, Molecular Neuroscience

Work in my laboratory focuses on the intracellular signaling mechanisms that regulate growth cone motility and guidance. Growth cones are sensory-motor specializations at the tips of developing axons and dendrites, which detect and transduce extracellular cues into guided extension. Guidance of growing axons to their proper synaptic targets sites serves a crucial early step in the development of specific synaptic connectivity. Great advances have been made in recent years in our understanding of the factors that contribute to guided axon extension. Many new classes of ligands and their receptors have been discovered and we are beginning to appreciate how growth cones integrate multiple extracellular stimuli and convert those signals into stereotyped behaviors. However, it is clear that the number of specific synaptic connections far exceeds the number of guidance cues and receptors that are expressed by neurons. Therefore, epigenetic mechanisms, such as biochemical signal cascades, must provide additional information that is required to organize the highly complex interconnections of the adult nervous system.

Research in my laboratory combines a variety of fluorescent probe technologies with confocal microscopy to visualize the dynamic behavior of living growth cones and assess their physiological responses during axon extension in vitro and guided outgrowth in the intact spinal cord or retinotectal pathway. We use the African Clawed frog Xenopus Laevis as our model system due to the large size, rapid development, and ease of molecular and surgical manipulation of its embryos. We use both spinal cord and retinal ganglion neurons for our studies as the former allows us to study a variety of neuronal types with distinct synaptic partners, while the later provides a relatively homogenous neuronal population. Molecular expression, gene knock-down as well as photolytic uncaging techniques are used to alter the physiology of growth cones both in vitro and in vivo. By combining the latest advances in imaging technologies with improved optical probes including fluorescent fusion proteins and FRET-based reporter molecules, we are addressing questions regarding the molecular basis of axon outgrowth and guidance.

Diffusible, cell surface and extracellular matrix associated guidance cues bind to cell-surface receptors on growth cones to promote adhesion and activate intracellular signaling cascades. One intracellular signal that has particularly diverse effects on axonal and dendritic growth is cytosolic calcium (Ca2+) ions. Transient elevations of intracellular Ca2+ in growth cones can promote, inhibit or orient motility depending on the size, duration and distribution of these signals, as well as on the downstream Ca2+-dependent targets available. Although Ca2+ signaling clearly has profound influences on the motility of variety of neuronal types from many species, we still have little mechanistic understanding of how Ca2+ exerts such diverse affects. One current focus of my laboratory is in the identification of novel plasma membrane calcium channels that are activated by mechanical stretch. We have evidence that Ca2+ influx through stretch-activated channels (SACs) slows outgrowth.

One particular type of Ca2+ signal we discovered more recently are short duration Ca2+ bursts that are localized to the tips of growth cone filopodia (Gomez et al, 2001). These local Ca2+ signals were found to immobilize filopodia through activation of the Ca2+-dependent protease calpain (Robles et al, 2003). These results suggest that at the tips of filopodia are proteins that function to promote filopodial extension and that these proteins are cleaved and inhibited by Ca2+/calpain signals. This work has opened a new focus of my lab as we seek to understand the regulation of actin polymerization at the tips of filopodia. Using a biosensor that detects Src kinase-dependent phosphotyrosine (PY) signals in live cells, we have found that tyrosine phosphorylated proteins are enriched at the tips of extending filopodia. We have identified several tyrosine phosphorylated proteins at the filopodial tip and are beginning to dissect the sequence of molecular events responsible for filopodial protrusion (Robles, et al., 2005).

As apart of our current and future goals we will use the highly tractable Xenopus model system for analysis of cell signaling mechanisms during axon guidance in vivo. We have developed techniques to manipulate signaling pathways and image developing spinal neurons in vivo. Using both live imaging and 3D reconstructions of immunolabelled spinal cords, we have already extensively characterized normal axon pathfinding by spinal commissural interneurons (Moon and Gomez, submitted). Future work will utilize fluorescent biosensors and cell signaling manipulations to understand the molecular basis of axon pathfinding decisions by a variety of neurons at specific choice points in vivo.

Website:

http://gomez.anatomy.wisc.edu/

Selected Publications:

• Jacques-Fricke, B.T., Y. Seow, P.A. Gottlieb, F. Sachs and T.M. Gomez. 2006. Opposing regulation of neurite growth by Ca2+ influx through a mechanosensitive channel and release from intracellular stores. J Neurosci. 26(21): 5656-5664.
• Woo, S.W. and T.M. Gomez. 2006. Rac1 and RhoA promote neurite outgrowth through formation and stabilization of growth cone point contacts. J. Neurosci. 26(5): 1418-1428..
• Gomez, T.M. and J. Zheng. The molecular basis for calcium-dependent axon pathfinding. Nat. Rev. Neurosci. 7: 115-125.
• Kholmanskikh, S.S., H.B. Koeller, A. Wynshaw-Boris, T.M. Gomez, P.C. Letourneau, and E.M. Ross. 2006. Calcium-dependent interaction of Lis1 with IQGAP1 and Cdc42 promotes neuronal motility. Nat. Neurosci. 9: 50-57. [PDF]
• Moon, M-s and T.M. Gomez. 2005. Adjacent pioneer commissural interneuron growth cones switch from contact avoidance to axon fasciculation after midline crossing. Dev. Biol. 288: 474-486.
• Robles, E., S. Woo, and T.M. Gomez. 2005. Coincident Src and Cdc42 signals at the tips of growth cone filopodia regulate protrusion. J. Neurosci. 25: 7615-7622.
• Gomez, T.M. 2005. Neurobiology: Channels for pathfinding. Nature. 434: 835-838.
• Gomez, T.M. and E. Robles. 2004. The great escape: Phosphorylation of Ena/VASP by PKA promotes filopoidal formation. Neuron. Apr 8;42(1): 1-3.
• Gomez, T.M., D. Harrigan, J. Henely, and E. Robles. 2003. Working with Xenopus spinal neurons in live cell culture. Meth. Cell Biol. 71: 130-154.
• Robles, E., A. Huttenlocher, and T.M. Gomez. 2003. Filopodial calcium transients regulate growth cone motility and guidance through local activation of calpain. Neuron 38: 597-609. [PDF]
• Gomez, T.M., and E. Robles. 2003. Imaging calcium dynamics in developing neurons. Meth. Enzymol. 361: 407-422.
• Gomez, T.M., E. Robles, M-m. Poo, and N.C. Spitzer. 2001. Filopodial calcium transients promote substrate-dependent growth cone turning. Science. 291: 1983-1987. [PDF]