Golli modulates oligodendroglial cell migration in vitro
Using time-lapse video microscopy we examined the effect of golli on OPC migration. These experiments were performed over a period of 24 hours on OPCs isolated from control, KO, and JOE mice, in medium containing PDGF and bFGF (10ng/ml). In this time-lapse two-dimensional cell migration assay, cell movement was assessed by calculating the average cell migration velocity and the total distance traveled by the cell. For this analysis, only OPCs moving more than 50 μm in 6 hours were scored. Tracking of cells was performed using the SlideBook™
4.1 data analysis program described in Materials and Methods. Migrating OPCs were automatically followed by tagging a color or number to each cell examined, which were then tracked from frame to frame. Examples of such measurements are shown in , in which four golli overexpressing cells are colored in green, red, yellow and blue. The easiest cell to track in this presentation is the green cell, which clearly moves a significant distance over the period examined. Movement of the other cells is less obvious but they were clearly measurable (See Supplementary video 1
). Under these experimental conditions the mean rate of migration for control and golli KO OPCs was 26 ± 4.5 μm/h and 18 ± 2.8 μm/h, respectively, P<0.01 (). So the average cell migration velocity in golli KO OPCs was significantly reduced compared with that of the control group. In similar experiments the average cell velocity in golli overexpressing cells (JOE) was found to be almost double that of the JOE control cells (48 ± 4.1 μm/h and 23 ± 3.7 μm/h, respectively, P<0.01) (). As might be expected, there was an increase in the total migration distance (), and we also found an increase in the number of migrating cells (cells moving more than 50 μm in 6 h) in the JOE OPC population ().
Overexpression of Golli enhances OPC migration
Further analysis of cell migration was performed using the Transwell system, which provides a three-dimensional assessment of motility. Counting of cells in these experiments was facilitated by use of OPCs isolated from golli KO and JOE mice that were bred onto a background in which OPCs are tagged with GFP (Mallon et al., 2002
). OPCs were plated on one side of the membrane and migrating, fluorescently tagged cells were counted on the other side of the membrane after 24 (1DIV), 48 (2DIV) and 72 hours (3DIV). In the presence of PDGF (20ng/ml) the fluorescently labeled golli overexpressing (JOE) cells were found to migrate faster than the control cells in this assay (). Conversely, golli-deficient OPCs were observed to migrate slower than control OPCs (). These findings are in good agreement with the direct measurement of velocity made in cultured OPCs. Overall, the data showed increased cell migration velocity and total migration distance as well as increased numbers of migrating cells in the JOE population.
Migrating cells move in a saltatory fashion
Migrating cells move in a saltatory fashion, alternating periods of higher and lower speed at a frequency of ~1–2 cycles/h. These cycles reflect the steps requires for directed OPC movement: extension of the leading process, translocation of the soma/nucleus (nucleokinesis), and retraction of the trailing process, these three individual steps constitute a single migration cycle (saltatory movement). We measured the average frequency and amplitude of saltatory movement of OPCs migrating in our culture system, and examples of this in control, KO and JOE OPCs are shown in . We found a significant decrease in the frequency of saltatory oscillations in KO OPCs compared to control cells (1.41 ± 0.20 cycles/h, and 2.12 ± 0.14 cycles/h, respectively, n=25 P<0.05) (). Additionally, the average maximum speed in KO OPCs was significantly lower than the average maximum speed in control cells (32 ± 3.1 μm/h and 41 ± 3.0 μm/h, respectively, n=20 P<0.05). These data suggest that the average speed changes in golli KO cells are due to a reduction in the maximum speed reached during the soma translocation together with an increase in the duration of resting phases of migration.
There was no difference between the frequency of these saltatory oscillations in JOE control and JOE cells (1.96 ± 0.21 cycles/h, and 2.01 ± 0.18 cycles/h, respectively, n=25) (). However, the average maximum speed in JOE OPCs was significantly higher than in the JOE control cells (67 ± 5.1 μm/h, and 38 ± 3.4 μm/h, respectively, n=25 P<0.01) (). These data indicate that the amplitude of saltatory oscillations (difference between maximum and minimum speeds during nucleokinesis), but not the resting times between cycles (frequency), is responsible for the greater migration rates of the JOE OPCs.
Using high resolution spatiotemporal microscopy, we determined the average length of individual leading processes in migrating OPCs before the initiation of the migration cycle (before nucleokinesis). We found that the average leading process was significant longer in JOE cells and significantly shorter in KO OPCs than in corresponding control cells, demonstrating that during OPC migration golli overexpression promotes leading process extension, (). Taken together these experiments localized the step in the migration process in which golli plays a role and suggest that golli modulates OPC migration by accelerating both nucleokinesis and leading process growth. Faster nucleokinesis could be responsible for the higher amplitude (difference between lowest and highest speed) found in JOE cells and slow leading process formation could be responsible for the increase in the resting time between cycles of advancement in golli KO OPCs.
Spontaneous Ca++ oscillations modulate OPC migration
We tested the possibility that the observed effects on OPC migration were due to the effects of golli on Ca++
uptake by performing live imaging experiments to examine and correlate cell mobility with intracellular Ca++
changes in primary cultures of OPCs isolated from golli KO and JOE mice. The combined use of real time confocal microscopy and Ca++
indicator dye (Fluo-4) reveals that OPCs exhibit transient Ca++
elevations as they migrate in vitro ( and Supplementary video 2
). The frequency and amplitude of Ca++
transients in the OPC somata changes dynamically during their migration (Supplementary Figure 1 and Figure 4B
) and it correlates positively with the rate of cell movement (correlation coefficient, 0.91 r2
). Interestingly, golli overexpression was associated with a significant increase in the mean Ca++
oscillation amplitude from 87.2 ± 2.2 nM in control cells to 117.2 ± 3.1 nM (p
<0.01) in JOE cells (
), an effect that is reflected in the rightward shift in the frequency distribution of spontaneous events in JOE versus JOE control OPCs shown in . This increase in amplitude of spontaneous Ca++
events could be caused by the addition of a few very large events or a shift in the size of all events. To investigate these two possibilities, we constructed cumulative probability histograms of spontaneous Ca++
oscillations amplitudes from JOE control and golli-overexpressing OPCs (). These two distributions were found to be significantly different (Kolmogorov Smirnov test, p
<0.001). The cumulative probability shown in is an average cumulative probability ± SEM from 18 cells for each genotype. Beginning at the third bin (30nM), the two cumulative probabilities are significantly different in each bin by the t
<0.05). This analysis suggests that the entire population of events is increased in size, with the median increasing by ~35%. On the other hand, the absence of golli was associated with a significant decrease in the mean Ca++
oscillation amplitude from 93.5 ± 3.1 nM in control cells to 58.7 ± 4.0 nM (p
), an effect that is reflected in the leftward shift in the frequency distribution of Ca++
oscillations in KO versus control OPCs (). The cumulative probability histogram suggests that the entire population of events is decreased in size by ~37%. (). No significant differences were observed in the mean Ca++
oscillation frequency in any of the cell populations studied (data not shown
). These results reveal a modulation of the amplitude of spontaneous Ca++
oscillations in golli KO and JOE cells, which is likely to be one of the factors involved in the alterations in OPC migration that we observed in cells lacking and overexpressing golli.
Spontaneous Ca++ transients in migrating OPCs
Golli stimulates the amplitude of Ca++ oscillation in oligodendroglial cells
Golli proteins play a role in modulating OPC migration through VOCCs
It was shown previously that golli proteins play a key role in the modulation of voltage-dependent Ca++
influx in OPCs (Paez et al., 2007
) and recent studies suggest that VOCCs generate Ca++
signals that play a vital role in the migration of cerebellar granule cells (Komuro and Rakic, 1992
). For this reason, we examined the role played by VOCCs on OPC migration by performing several combined Ca++
imaging/cell migration experiments in the presence of pharmacological agents to stimulate or inhibit voltage-gated Ca++
uptake in OPCs. First, we examined the effect of lowering extracellular Ca++
levels through chelation with EGTA or by reducing the [Ca++
] in the medium. Second, we assessed the effect of specific L-type VOCC blockers such as nifedipine and verapamil. These treatments resulted in a significant reduction in the Ca++
transient frequency and amplitude and a slowdown of OPC movement (), indicating that VOCCs, known to contribute to homeostatic Ca++
balance in OPCs and other cells, are important in modulating OPC migration. Of considerable interest, is that stimulation of Ca++
influx through the voltage-gated Ca++
channels (through high K+
treatment) significantly increased the Ca++
transient frequency and amplitude and accelerated cell movement (). Similar results were found using Bay K 8644, an L-type Ca2+
channel agonist which prolongs single channel open time without affecting the close time (). These data show that changes in Ca++
transients resulting from the modulation of voltage-gated Ca++
influx provide a powerful means by which OPC migration may be regulated in vitro. These results also demonstrate that OPC movement is related to the frequency and amplitude of Ca++
transients in OPC somata, and that Ca++
transient frequency and amplitude provides an intracellular signal for controlling the rate of OPC migration.
Effects of the changes in the Ca ++ transient frequency and amplitude on migrating oligodendroglial cells
The above results show that spontaneous Ca++ oscillations in OLs are generated in response to voltage-dependent calcium channel activation. To investigate their role in golli-dependent modulation of migration velocity we tracked control and golli-overexpressing OPCs in medium containing the VOCC antagonist verapamil. shows that the average speed of OPC migration was lower in both JOE control and JOE OPCs when verapamil was present in the media. For example, in control media (Basal), the maximum average migration speed of JOE cells, was 67 ± 5.1 μm/h (n=28), but as the concentration of verapamil was increased, it fell to an average speed of 32 ± 2.6 μm/h (n=25) in the presence of 10 μM verapamil (). In 20 μM verapamil there was essentially complete inhibition of JOE cell migration (). In the same migrating cells, this treatment also resulted in a significant reduction in the Ca++ transient amplitude and frequency (). Similar results were found using nifedipine, another specific L-type VOCC blocker ().
VOCCs are essential for enhanced migration of JOE cell
In contrast, addition of high K+ to the medium, a manipulation that activates VOCCs by depolarizing the plasma membrane, there was an increase in the average cell velocity () as well as the amplitude of Ca++ transient in control and golli overexpressing cells (). In basal conditions, JOE cells migrated at an average rate of 48 μm/h with an average Ca++ transient amplitude of 117.2nM. In the presence of 15mM K+, JOE OPCs migrated at a significantly higher rate of 74 μm/h with an increased Ca++ transient amplitude of 128.4nM. Thus, high K+ increased the rate of cell movement in JOE OPCs, along with increasing the amplitude of Ca++ transients. Importantly, under this experimental condition (high K+), the migration speed and the amplitude of Ca++ transients observed in migrating JOE cells were significantly higher than those observed in control OPC (). Furthermore, potassium and golli-mediated modulation of OPC velocity disappeared when the VOCC antagonist verapamil was added to the external medium (). These results clearly indicate that the golli-induced acceleration of OPC movement may result from an increase in the amplitude of Ca++ transients generated by VOCCs.
VOCCs modulate the rate of OPC migration
In parallel time-lapse experiments, the effect of VOCCs inhibitors and high K+ was evaluated in migrating OPCs obtained from golli KO and control mice. As expected, we found a significant decrease in the average speed and in the amplitude of Ca++ transient induced by high K+ in KO cells vs control OPCs (). Additionally, the effect of golli ablation on OPC velocity disappeared when the VOCC antagonists verapamil and nifedipine were added to the medium ( and ). Changes in the frequency and amplitude of saltatory movements and Ca++ transients in golli KO and overexpressing OPCs are summarize in .
Changes in the frequency and amplitude of saltatory movements and Ca++ transients in golli KO and overexpressing OPCs under basal conditions or after the addition of 15mM K+ to the culture medium.
Perturbations of golli structure exert similar effects on OPC migration and Ca++ uptake
In mouse, three golli products have been identified: BG21, J37, and TP8 (Campagnoni et al., 1993
). In order to identify any motifs on the golli protein that might be important in the effects of golli on OPC migration we prepared mutated/deleted versions of J37 and BG21 fused to GFP. The GFP-mutated golli plasmids were transfected into the immature oligodendroglial cell line N19 (Verity et al., 1993
) and cell migration measured to define sites on the molecule that might be important in golli regulation of OPC migration. diagrams the mutations/deletions used for analysis. Using the agarose drop migration assay we found that elimination of the first 45 or 110 amino acids from the N-terminus of J37 (J37 Del1 and J37 Del2 respectively) completely obliterated the average cell velocity increase in the golli overexpressing cells (). Previously, Feng et al. (2006)
found that myristoylation of golli BG21 was important for targeting golli to the plasma membrane of Jurkat T-cells, and we found that mutation of the myristoylation sites of either golli J37 or BG21 (J37 and BG21 Myr) completely reversed the effects of golli on Ca++
uptake in N19 cell line (), indicating that membrane association is essential for golli action on the enhancement of Ca++
influx in OPCs (Paez et al. 2007
). As shown in , myristoylation of golli and, indeed the first 110 N-terminal amino acids, are essential for golli effects on cell migration, adding further evidence implicating a clear relationship between golli, Ca++
uptake and OPC migration.
The golli myristoylation site is essential for the effect of golli on OPC migration
Migration of subventricular zone OPCs is enhanced in golli overexpressing mice
We tested whether increased levels of golli enhanced OPC migration in vivo by time-lapse imaging of live tissue sections containing GFP-labeled OPCs in golli KO and JOE mice. These experiments were performed using a double transgenic mouse created by breeding the golli KO and JOE mice with a line expressing GFP under control of the PLP promoter (Mallon et al., 2002
). In these mice GFP expression provided a convenient marker for cells in the oligodendroglial lineage, thus facilitating the imaging experiments. We performed our in vivo measurements of OPC migration in slice preparations containing the lateral ventricle subventricular zone (SVZ) and corpus callosum since these regions have been well studied as sources of migrating OPCs. Our goal was to confirm the in vitro data with respect to rates of migration of OPCs out of the ventricular zone and into the corpus callosum using conditions established by others for such studies (Kakita and Goldman, 1999
; Suzuki and Goldman, 2003
). We tracked cell bodies of migrating GFP positive OPCs (OPC~GFP) for a period of 12 hours in the SVZ. In these time-lapse experiments cell movement was assessed by calculating the average cell migration velocity and the total distance traveled by the cell. Examples of such measurements are shown in . Under these experimental conditions the mean rates of migration for JOE control and JOE OPCs in the SVZ were 27 ± 3.4 μm/h and 43 ± 3.1 μm/h, respectively (p
<0.01). So the in vivo average cell migration velocity in golli overexpressing OPCs was significantly higher compared with that of the control group (). As might be expected, there was also an increase in the total migration distance after 8 hours (JOE Control: 180 ± 37 μm, and JOE: 274 ± 57 μm, n
=30 cells, P<0.01). Conversely, a reduced migration velocity compared to controls was noted in golli KO cells, where OPCs lacking golli appeared to migrate slower than control OPCs in vivo (). Furthermore, and clearly indicating that VOCCs are essential for in vivo OPC migration, 15mM K+
caused a significant increase in the average cell velocity in golli-overexpressing OPCs (), and in both genotypes a complete inhibition of OPC migration was found in the presence of VOCC inhibitors ().
Migration of subventricular zone OPCs in living tissue
A model for the data presented in this work is proposed in . In the absence of golli, there is a significant decrease in the amplitude of Ca++ transients as well as in the average frequency and amplitude of saltatory movement of migrating OPCs. In contrast, golli overexpression enhances activation of L-type VOCCs leading to increases in the amplitude of Ca++ transients and accelerating OPC migration by promoting Ca++ dependent soma translocation and leading process extension. It is not yet clear whether golli acts in a direct or indirect fashion on the channel itself.