We are interested in studying the cellular mechanisms of calcium signalling by glial cells in the nervous system. Glial cells and neurons are in intimate communication with each other during central nervous system (CNS) development and normal brain function. Glial cells monitor and respond to neural activity by conditioning the extracellular milieu, signalling within glial cell networks, and sending signals back to neurons. Such signalling takes the form of propagated Ca2+ waves that spread over long distances in response to synaptic activity. One of our objectives is to understand the processes that support temporal and spatial characteristics of Ca2+ signals within and between cells. Another objective is to understand the precise nature of glial cell signals in response to neuronal activity and the consequence of such signals to CNS function.

Ca2+-Signalling Microdomains
Yagodin, Sheppard, Simpson
Work on astrocytes and oligodendrocyte progenitors (OP cells) in culture revealed that wave propagation is saltatory. Such propagation is achieved by assembling a number of highly specialized Ca2+-signalling microdomains that function to produce a discretely localized spurt of cytoplasmic Ca2+ increase.

Such focal signals regulate locally discrete cellular processes and support regenerative propagation of calcium waves through the entire cell and its processes. Ca2+-signalling microdomains are found five to seven micrometers apart along cellular processes and are characterized by high-density patches of endoplasmic reticulum (ER) proteins such as the inositol 1,4,5-trisphosphate receptors (IP3Rs), sarco-endoplasmic reticulum calcium pumps, calreticulin, and at least one mitochondrion in close association. The specialization of Ca2+ signalling microdomains allows for enhanced Ca2+ release at the sites. In addition to supporting long-distance wave propagation, the specialized sites provide for locally discrete Ca2+ signals that last only for brief periods (Fig. 16).

Figure 16

Ca2+ signaling microdomains

Local Ca2+ Release, Ca2+ Sparks
Haak
Using confocal microscopy, we recently completed a functional characterization of the signalling rafts in OP cell processes. Our aim was to measure the kinetics of elementary Ca2+ release events, Ca2+ sparks, and Ca2+ puffs (Fig. 17), which are the smallest units of local Ca2+ release and presumed to emanate from clusters of IP3Rs on the ER.

Two different observations suggested that IP3Rs and RyRs may modulate each other and thereby influence the local Ca2 signal. Blockade of RyRs with ryanodine increased the amplitude and width of IP3R-mediated puffs, pointing to a larger flux through the channels or activation of a greater number of channels. Conversely, after exposure of cells to MeCh, potentiated RyRs and DMPX-induced events increased in size. The mechanism of such modulation is currently unknown. It is likely that the two channel types may be expressed in patches, and their proximity to each other and sensitivity to ambient Ca2+ concentration may be modulated by the activity of the other.

We have focused on glial cell processes, particularly cells of the oligodendrocyte lineage and astrocytes. We propose to undertake a molecular characterization of these specialized Ca2+ release sites to determine the interacting protein assemblies that orchestrate this process. In addition, we propose to probe the functional significance of unique proteins found in the macromolecular assembly. We will investigate if the microdomains are preferentially localized to predestined cellular sites of contact to support optimal signalling between neurons and glial cells and between glial cells.

The goal is to isolate intact oligomeric protein assemblies that make up specialized microdomains of Ca2+ signalling from glial cell membranes isolated from mouse brains. The enriched glial cell membrane preparations will be used in an affinity separation strategy to obtain macromolecular assemblies. We will use modern proteomic technologies for molecular identification of component protein machines that make up the specialized Ca2+ release microdomains. The technology for proteomics involves separation of protein components in the macromolecular assembly followed by modern molecular identification using spectrometry. We will separate proteins by 1D and 2D PAGE and identify spots by staining before excising them for digestion with trypsin. Peptide pools in the tryptic digests will be identified with mass spectrometry by using MALDI-TOF. We will compare peptide mass data with protein databases for positive hits. Tandem LC-MS/MS is another technique that will allow for peptide mass determination and database comparison. We will select interesting novel proteins present in the microdomains to investigate cellular localization and functional importance.