CURRENT PROJECTS

Role of myosin II in dendrite and axon formation

We are investigating the role of myosin II in the extension and formation of dendrite precursors from cultured embryonic forebrain neurons. In addition, we are addressing the roles of upstream myosin II regulatory kinases (RhoA-kinase and Myosin Light Chain Kinase) in the regulating myosin II activity and thus dendrite and axon formation. The ultimate goal of this project is to define the functions of myosin II and its upstream regulatory kinases at all stages of dendrite and axon formation (Kollins).

Example of the stages of forebrain neuron morphologic differentiation in vitro. Neurons are stain against F-actin (red) and microtubules (green). Stage I neurons exhibit short F-actin rich filopodial/lamellipodial protrusions. Stage II neurons develop multiple short processes, termed minor processes, that exhibit F-actin accumulations at their tips and are supported by a microtubule bundle. Stage II neurons continue to exhibit minor processes, but one of these processes has extended much longer than the other minor processes and has become the axon.

Role of PI3K in axon maintenance and retraction

PI3K is an enzyme that phosphorylates lipids and proteins. Inhibition of PI3K in sensory neurons results in growth cone collapse and axon retraction. We are investigating the downstream signaling and cytoskeletal events that cause axon retraction in response to inhibition of PI3K (Orlova).

Example of axon retraction in response to pharmacological inhibition of PI3K. Time in minutes is shown prior to (-2 to 0 min) and after inhibition (+5-+20 min).

Regulation of myosin II isoform distribution in growth cones

        Myosin II has two major isoforms, IIA and IIB, with different functional properties. We are currently investigating how extracellular signals regulate the sub-cellular distribution of these two isoforms in the growth cones of embryonic sensory axons. Myosin II isoforms are being detected using isoform specific antibodies. The contributions of extracellular matrix molecules and intracellular signaling pathways are under investigation (Ketschek).

Actin dynamics in axons

Actin filaments drive cell motility (i.e., protrusion of filopodia and lamellipodia). We are investigating the dynamics of actin in axons and growth cones transfected to express EGFP-actin. By directly imaging actin dynamics we seek to determine the effects of guidance cues on the cytoskeletal dynamics underlying axon guidance. Through a combination of live imaging of actin dynamics and experimental treatments we seek to directly link specific signaling pathways to features of actin dynamics in living growth cones and axons. (Loudon, Gallo)

Example of EYFP-beta actin transfected DRG growth cone. Numbers reflect seconds in sequence.

Axonal transport of cytoskeletal proteins

Cytoskeletal proteins, tubulin and actin, are synthesized in the cell body and transported down the axon to growth cones and synapses. The issue of the form of the cytoskeletal proteins undergoing transport (i.e., soluble or polymeric) is a controversial issue. We are using combinations of live imaging of fluorescent proteins and experimental interventions to investigate this issue. By combining multiple approaches including photobleaching and photoactivation of fluorescent proteins we hope to gain additional data to assist in the determination of the form of cytoskeletal protein transport. (Gallo, Jones).

Example of an EGFP-actin expressing distal axon that was photobleached (PHB). By 30 min post PHB approximately 40% of the EGFP-actin signal has recovered from PHB, reflecting the transport of actin from the proximal axon into the PHB distal axon.

Rho-family GTPases as regulators of axon extension

Rho-family GTPases (RhoA, Rac1 and Cdc42) control actin filament organization and dynamics. We are investigating the specific roles of these proteins and their downstream effectors (e.g., PAK1 and Rho-kinase) in axon extension and guidance. Using the yeast-two-hybrid method we are developing specific binding mutants of GTPases that will assist in the experimental dissection of the molecular components of these signaling pathways. (Loudon, Silver)

Magnetic patterning of cells

We are collaborating with engineers (Dr. G. Friedman and Dr. K. Barbee, Drexel University) to develop a method to pattern multiple cell types on the same substratum (i.e., control the position of individual cell bodies en mass). The ultimate goal of this research is to create defined neuronal circuits in vitro. However, the ability to control cell positioning in vitro may have applications in biotechnology and the design of experimental paradigms. In order to pattern cells we attached paramagnetic particles to cell bodies and directed their deposition by the localized application of magnetic fields generated by patterns on the substratum. For additional work on the use of magnetism in biological applications see the Magnetic Microsystems Group webpage. (Francisco)

Example of patterned DRG cells 24 hrs in vitro (neurons, Schwann cells and fibroblasts). The magnetic patterns in this example were V shaped. The cells were stained with a membrane dye to reveal morphology. The red lines indicate the location of the magnetic pattern. By this time neurons extended axons emanating from the cell bodies positioned on the magnetic patterns.

 

An additional example of cells, labeled red with a fluorescent dye, that have been patterned in an array. The horizontal green lines represent the magnetic pattern. Note all the cells are deposited at only one end of the magnetic strips, the end that had been magnetized.

 

Biomechanics of Axon Retraction

Axon retraction in response to semaphorin 3A requires myosin II activity and an axonal F-actin bundle cytoskeleton (JCS, 119(16):3413). We are continuing work on further understanding the biomechanics of axon retraction. Using Atomic force Microscopy we seek to measure axon stiffness and force generation during axon retarction. (Kulkarni: Collaboration with Dr. K. Barbee, see link above)

Atomic Force Microscopy Image of  a growth cone.

Developmental changes in the mechanism of axon extension

We are investigating differences in the cytoskeletal basis of axon extension between early and late embryonic neurons. This is project is a collaboration with Dr. M. Selzer (U. of Pennsylvania). Previous work has shown that during development sensory neurons change their pattern of axon extension from a few long axons to multiple shorter axons. In order to further determine how the mechanism of axon extension changes during development, we are testing the contribution of individual cytoskeletal components and regulatory proteins at different developmental ages of sensory neurons. (Jones, Ketscheck)

 

Following a glitch in the matrix.... Steve Jones presented his poster...

(digital manipulation credits to Herb Francisco)

a visitor discusses one of the posters with us.

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