Phosphatidylinositol 3-kinase facilitates microtubule-dependent membrane transport for neuronal growth cone guidance

Abstract

The activity of PI3K is necessary for polarized cell motility. To guide extending axons, environmental cues polarize the growth cone via asymmetric generation of Ca(2+) signals and subsequent intracellular mechanical events, including membrane trafficking and cytoskeletal reorganization. However, it remains unclear how PI3K is involved in such events for axon guidance. Here, we demonstrate that PI3K plays a permissive role in growth cone turning by facilitating microtubule (MT)-dependent membrane transport. Using embryonic chick dorsal root ganglion neurons in culture, attractive axon turning was induced by Ca(2+) elevations on one side of the growth cone by photolyzing caged Ca(2+) or caged inositol 1,4,5-trisphosphate. We show that PI3K activity was required downstream of Ca(2+) signals for growth cone turning. Attractive Ca(2+) signals, generated with caged Ca(2+) or caged inositol 1,4,5-trisphosphate, triggered asymmetric transport of membrane vesicles from the center to the periphery of growth cones in a MT-dependent manner. This centrifugal vesicle transport was abolished by PI3K inhibitors, suggesting that PI3K is involved in growth cone attraction at the level of membrane trafficking. Consistent with this observation, immunocytochemistry showed that PI3K inhibitors reduced MTs in the growth cone peripheral domain. Time-lapse imaging of EB1 on the plus-end of MTs revealed that MT advance into the growth cone peripheral domain was dependent on PI3K activity: inhibition of the PI3K signaling pathway attenuated MT advance, whereas exogenous phosphatidylinositol 3,4,5-trisphosphate, the product of PI3K-catalyzed reactions, promoted MT advance. This study demonstrates the importance of PI3K-dependent membrane trafficking in chemotactic cell migration.

FIGURE 1.

Ca²⁺-induced growth cone attraction depends on PI3K activity. A, time-lapse DIC images showing growth cone responses to attractive Ca²⁺ signals. In the absence (upper panels) or presence (lower panels) of LY294002, Ca²⁺ signals were generated on one side of the growth cone (white spots) by FLIP of caged Ca²⁺ every 3 s. Digits represent minutes after the start of repetitive FLIP. Scale bar = 10 μm. B, growth cone responses to attractive Ca²⁺ signals generated by FLIP of either caged Ca²⁺ or caged IP₃ in the absence (control) or presence of the indicated drugs. Positive and negative values on the x axis indicate attractive and repulsive turning, respectively. Numbers in parentheses indicate the number of growth cones examined. *, p < 0.05 (Dunnett's multiple comparison test). C, control or Akt-PH-EGFP-expressing growth cones were tested for their turning responses to FLIP of caged Ca²⁺. Numbers in parentheses indicate the number of growth cones examined. *, p < 0.05 (unpaired t test).

FIGURE 2.

CICR-elicited centrifugal vesicle transport depends on PI3K activity. A, fluorescent and DIC images of an FM1-43-loaded growth cone. Ca²⁺ signals were generated every 3 s by repetitive UV photolysis of caged Ca²⁺ at the area denoted by yellow circles. Arrowheads indicate FM1-43-labeled vesicles that migrated centrifugally on the side with Ca²⁺ signals (near side). Blue lines depict the growth cone outline. Digits represent seconds after the start of UV photolysis. Scale bars = 5 μm. B, frequency of centrifugal vesicle migration on the near and far sides of the growth cone before (Pre) and after (UV) the start of repetitive UV irradiation. Caged Ca²⁺ loading was omitted in Blank. Numbers in parentheses indicate the number of growth cones examined. The Bonferroni multiple comparison test was used to compare 1) the frequency between both sides of the growth cone and 2) the frequency before and after photolysis on each side. #, p < 0.05 (near side versus far side during UV photolysis); *, p < 0.05 (Pre versus UV on the near side).

FIGURE 3.

IICR elicits centrifugal vesicle transport in a PI3K-dependent manner. A, fluorescent and DIC images of an FM1-43-loaded growth cone. Ca²⁺ signals were generated every 3 s by repetitive UV photolysis of caged IP₃ at the area denoted by yellow circles. Arrowheads indicate FM1-43-labeled vesicles that migrated centrifugally on the side with Ca²⁺ signals (near side). Blue lines depict the growth cone outline. Digits represent seconds after the start of UV photolysis. Scale bars = 5 μm. B, frequency of centrifugal vesicle migration on the near and far sides of the growth cone before (Pre) and after (UV) the start of repetitive UV irradiation. Numbers in parentheses indicate the number of growth cones examined. The Bonferroni multiple comparison test was used to compare 1) the frequency between both sides of the growth cone and 2) the frequency before and after photolysis on each side. ##, p < 0.01 (near side versus far side during UV photolysis); *, p < 0.05 (Pre versus UV on the near side).

FIGURE 4.

PI3K regulates MT distribution in the P-domain of growth cones. A, immunofluorescent images showing MT distribution in growth cones treated with dimethyl sulfoxide (DMSO), LY294002, or wortmannin. Yellow dashed lines represent the C-domain/P-domain boundary. Scale bar = 5 μm. B–D, effect of PI3K inhibitors on the total length and number of MTs (B and C, respectively) and the length of individual MTs (D) in the P-domain of growth cones. The value of each growth cone was normalized to the mean of control growth cones. The graphs show pooled data of three independent experiments. Numbers in parentheses indicate the total number of growth cones examined. **, p < 0.01 versus the control (Dunnett's multiple comparison test).

FIGURE 5.

Visualization of the actively polymerizing plus-ends of MTs. Shown are time-lapse fluorescent images of an EB1-EGFP-expressing growth cone before (A) and after (B) treatment with LY294002. Digits represent time (minutes:seconds). LY294002 was applied to the medium at ∼0 min. Scale bar = 5 μm. Magnified views of regions of interest (ROI) are shown.

FIGURE 6.

PI3K facilitates MT advance into the growth cone P-domain. Shown is the effect of PI3K inhibitors, PIP₃, and PIP₂ on EB1 dynamics in growth cones. As controls, the growth cones were treated with vehicles: dimethyl sulfoxide (DMSO) for PI3K inhibitors and H₂O for PIP₃ and PIP₂. A, the number of EB1 comets that crossed the unit length of the C-domain/P-domain boundary was compared in each growth cone before (Pre) and after (Post) drug treatment. Each line represents a drug-induced change in a single growth cone. B and C, shown are the drug-induced changes in the life time (B) and speed (C) of EB1 comets that had crossed the boundary. *, p < 0.05 (paired t test); **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Inhibition of the PI3K signaling pathway attenuates MT advance into the growth cone P-domain. The following parameters of EB1-mCherry dynamics were compared between control and Akt-PH-EGFP-expressing growth cones: the number of EB1 comets that crossed the unit length of the C-domain/P-domain boundary (A), the life time of EB1 comets (B), and the speed of EB1 comets (C). Numbers in parentheses indicate the number of growth cones examined. **, p < 0.01 (unpaired t test); ***, p < 0.001.