Biomedical subjects

Gary A Banker

Publications and source records attributed to Gary A Banker.

5 recordsLinked to original sources

PubMed2006

Effects of substrate geometry on growth cone behavior and axon branching.

At the leading edge of a growing axon, the growth cone determines the path the axon takes and also plays a role in the formation of branches, decisions that are regulated by a complex array of chemical signals. Here, we used microfabrication technology to determine whether differences in substrate geometry, independent of changes in substrate chemistry, can modulate growth cone motility and branching, by patterning a polylysine grid of narrow (2 or 5 microm wide) intersecting lines. The shape of the intersections varied from circular nodes 15 microm in diameter to simple crossed lines (nodeless intersections). Time-lapse recordings of cultured hippocampal neurons showed that simple variations in substrate geometry changed growth cone shape, and altered the rate of growth and the probability of branching. When crossing onto a node intersection the growth cone paused, often for hours, and microtubules appeared to defasciculate. Once beyond the node, filopodia and lamellipodia persisted at that site, sometimes forming a collateral branch. At nodeless intersections, the growth cone passed through with minimal hesitation, often becoming divided into separate areas of motility that led to the growth of separate branches. When several lines intersected at a common point, growth cones sometimes split into several subdivisions, resulting in the emergence of as many as five branches. Such experiments revealed an intrinsic preference for branches to form at angles less than 90 degrees . These data show that simple changes in the geometry of a chemically homogeneous substrate are detected by the growth cone and can regulate axonal growth and the formation of branches.

Animals↗

PubMed2006

Activated c-Jun N-terminal kinase is required for axon formation.

A critical transition in neuron development is formation of the axon, which establishes the polarized structure of the neuron that underlies its entire input and output capabilities. The morphological events that occur during axonogenesis have long been known, yet the molecular determinants underlying axonogenesis remain poorly understood. We demonstrate here that axonogenesis requires activated c-Jun N-terminal kinase (JNK). JNK is expressed throughout the neuron, but its phosphorylated, activated form is highly enriched in the axon. In young axons, activated JNK forms a proximodistal gradient of increasing intensity, beginning at about the point where the axon exceeds the lengths of the other neurites (minor processes). Treatment with SP600125, a specific inhibitor of JNK, reversibly inhibits axonogenesis but does not prevent the formation of minor processes or their differentiation into dendrites (based on their immunostaining with marker proteins). Expression of a dominant-negative construct against JNK similarly prevents axonogenesis. Investigation of JNK targets revealed that activating transcription factor-2 is phosphorylated under normal conditions in neurons, and its phosphorylation is significantly attenuated after JNK inhibition. These results demonstrate that activated JNK is required for axonogenesis but not formation of minor processes or development of dendrites.

Activating Transcription Factor 2↗

PubMed2006

The role of protein interaction motifs in regulating the polarity and clustering of the metabotropic glutamate receptor mGluR1a.

When expressed in cultured hippocampal neurons, the metabotropic glutamate receptor mGluR1a is polarized to dendrites and concentrated at postsynaptic sites. We used a mutational analysis to determine how previously identified protein interaction motifs in the C terminus of mGluR1a contribute to its localization. Our results show that the polyproline motif that mediates interaction with Homer family proteins is critical for the synaptic clustering of mGluR1a. A single point mutation in this motif, which prevents the binding of Homer with mGluR1a, reduced its colocalization with a postsynaptic marker to near-chance levels but did not affect its dendritic polarity. In contrast, deleting the PDZ (postsynaptic density-95/Discs large/zona occludens-1) binding domain, which interacts with Tamalin and Shank, had no effect on synaptic localization. Neither of these protein interaction motifs is important for trafficking to the plasma membrane or for polarization to dendrites. Although deleting the entire C terminus of mGluR1a only modestly reduced its dendritic polarity, this domain was sufficient to redirect an unpolarized reporter protein to dendrites. These observations suggest that mGluR1a contains redundant dendritic targeting signals. Together, our results indicate that the localization of mGluR1a involves two distinct steps, one that targets the protein to dendrites and a second that sequesters it at postsynaptic sites; different protein interactions motifs mediate each step.

Animals↗

PubMed2006

A change in the selective translocation of the Kinesin-1 motor domain marks the initial specification of the axon.

We used the accumulation of constitutively active kinesin motor domains as a measure of where kinesins translocate in developing neurons. Throughout development, truncated Kinesin-3 accumulates at the tips of all neurites. In contrast, Kinesin-1 selectively accumulates in only a subset of neurites. Before neurons become polarized, truncated Kinesin-1 accumulates transiently in a single neurite. Coincident with axon specification, truncated Kinesin-1 accumulates only in the emerging axon and no longer appears in any other neurite. The translocation of Kinesin-1 along a biochemically distinct track leading to the nascent axon could ensure the selective delivery of Kinesin-1 cargoes to the axon and hence contribute to its molecular specification. Imaging YFP-tagged truncated Kinesin-1 provides the most precise definition to date of when neuronal polarity first emerges and allows visualization of the molecular differentiation of the axon in real time.

Animals↗

PubMed2003

Patterning axonal guidance molecules using a novel strategy for microcontact printing.

We present here a two-step strategy for micropatterning proteins on a substrate to control neurite growth in culture. First, conventional microcontact printing is used to prepare a micropattern of protein A, which binds the Fc fragment of immunoglobulins. Then, a chimeric protein, consisting of the extracellular domain of a guidance protein recombinantly linked to the Fc fragment of IgG (prepared using conventional molecular techniques), is applied from solution. The chimeric protein binds to the patterned protein A, taking on its geometric pattern. Using this method, we have micropatterned the extracellular domain of the cell adhesion molecule, L1 (as an L1-Fc chimera) and demonstrated that it retains its ability to selectively guide axonal growth. L1-Fc micropatterned on a background of poly-L-lysine resulted in selective growth of the axons on the micropattern, whereas the somata and dendrites were unresponsive. Substrates bearing simultaneous micropatterns of L1-Fc and poly-L-lysine on a background of untreated glass were also created. Using this approach, cell body position was controlled by manipulating the dimensions of the poly-L-lysine pattern, and the dendrites were constrained to the poly-L-lysine pattern, while the axons grew preferentially on L1-Fc. The two-step microcontact printing method allows preparation of substrates that contain guidance proteins in geometric patterns with resolution of approximately 1 microm. This method should be broadly applicable to many classes of proteins.

Axons↗