Current Major Research Directions
Neurons display a complex architecture
that underlies their ability to integrate signals and transmit them
over long distances. In particular, neurons elaborate morphologically
and functionally distinct domains, such as axons, dendrites, synapses,
Nodes of Ranvier, and axonal initial segments (see diagram below) in
order to carry out their signaling functions. Different functional
domains differ in the membrane proteins that are displayed on their
surfaces. This segregation of membrane proteins to different domains
enables the neuron to grow out axons and dendrites using specific cell
adhesion molecules (such as L1), and to ultimately elaborate synapses
and signal vectorially. Proper functioning of neurons, therefore,
depends on correctly targeting a large number of proteins to specific
cellular sites of action. Incorrect protein targeting has been linked
to a variety of disease states and abnormalities, including
neurological disorders. My lab studies how vertebrate neurons assemble
and maintain the distinct plasma membrane domains that underlie
neuronal function with a particular focus on axons and axon initial
segments (see reviews by our laboratory: Lasiecka et al,
2009; Yap and Winckler,
2008; Winckler, 2004).
Our long-term goal is to understand the elaboration of neuronal
architecture on a molecular level and its disturbance in disease
states. Currently, we are characterizing the cellular mechanisms and
molecular regulation of polarized membrane transport in neurons,
focusing on the L1 CAM family of adhesion receptors, L1/NgCAM and
Neurofascin. These two major lines of inquiry are described in more
Distinct functional domains with distinct protein composition need to be elaborated and maintained in vertebrate neurons for proper neuronal functioning (from Yap and Winckler, 2008; image by: Tatiana Boiko). Different colors designate different domains containing different proteins.
Targeting of Neurofascin to the axon initial segment
Neurofascin is a member of the L1 family of adhesion receptors and localizes to axonal initial segments (AIS) and Nodes of Ranvier, sites where action potentials are generated. The axon initial segment is of crucial importance for neuronal function since it influences neuronal excitability. The accumulation of neurofascin at axon initial segments occurs even in cultured neurons which lack myelination. The yellow staining in thefigure is neurofascin and its binding partner ankyrinG accumulated at the axon initial segment. The dendrites are stained in blue (Image by: Tatiana Boiko). Current models ofhow the axon initial segment assembles are primarily based on steady-state analysis. We, and others, have demonstrated that binding to ankyrinG is necessary for correct steady-state localization of neurofascin to the axon initial segment (see our paper: Boiko et al., 2007). While important insights have been gained from steady-state analysis, new approaches are necessary in order to elucidate the cellular pathways and molecular mechanisms responsible for proper assembly of the axon initial segment. We are therefore combining steady state analysis with kinetic analysis, endocytosis assays, interference approaches, and live imaging to study the dynamics of axon initial segment assembly.
Targeting of L1/NgCAM to the axon
We showed in previous work that the axonal cell adhesion molecule L1/NgCAM requires trafficking through somatodendritic endosomes to reach the axon, i.e. it appears to reach the axon indirectly by transcytosis (see our papers: Wisco et al., 2003 and Yap et al., 2008). Other cargos, such as transferrin (Tfn) receptors and AMPA receptors, also traverse somatodendritic endosomes, but are not ultimately sorted to axons. We are now elucidating the subcellular organization and molecular regulation of polarized membrane transport in neurons, focusing in particular on endosomes. Several efforts are underway in our laboratory to determine which compartments and which regulators accomplish the task of sorting different proteins from somatodendritic endosomes to either axons or dendrites.
We have identified the neuronal early endosomal protein NEEP21 as a critical regulator for sorting L1/NgCAM from the early endosome to the recycling endosome (see our paper: Yap et al, 2008). Importantly, the NEEP21-positive early endosomes are distinct from the canonical EEA1-positive early endosomes and might represent a neuronal adaptation of the endosomal pathway. Surprisingly, Tfn does not significantly accumulate in NEEP21-endosomes. We are determining how trafficking through EEA1- and NEEP21-endosomes differs for NgCAM and Tfn, and how the two compartments relate to each other in time and space. We are also investigating the roles of syntaxin13, a protein found in a complex with NEEP21, and of the EHD family of proteins. Again, we are combining steady state analysis with kinetic analysis, endocytosis assays, interference approaches, and live imaging in cultured hippocampal neurons.