Filopodial Calcium Transients Promote Substrate-Dependent Growth Cone Turning T.M. Gomez, E. Robels, M. Poo, N.C. Spitzer

Supplementary Material

Supplemental Figure 1. Localized filopodial Ca²⁺ transients are generated at sites of b1 integrin clusters. (A) Pseudocolored maximum projection of a 30-s time-lapse sequence encompassing 240 confocal images of a Fluo-4 loaded growth cone. Bright regions indicate areas of Ca²⁺ transient activity. (B) The same growth cone now labeled for b1 integrin shows that regions of transient activity occur at sites of b1 integrin clusters. (C) Quantification of fluorescence along three filopodia (between colored arrowhead pairs in (A) identifies discrete regions of elevated Ca²⁺. Line scans were normalized to the individual baseline fluorescence of each filopodium, showing that increased fluorescence is not due to loading or volume differences. Numbers indicate the individual transients generated locally within each filopodium. (D) Magnified regions from (A) and (B) show colocalization of Ca²⁺ signals and integrin clusters. Bar in (A) and (B), 2 mm; bar in (D), 0.8 mm.

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Supplemental Figure 2. Global growth cone Ca²⁺ transients regulate turning. (A) The frequency of global growth cone Ca²⁺ transients increases as growth cones on LN approach a boundary with TC. At >40 mm from the border, the frequency of growth cone transients is similar to that on LN alone, but increases as growth cones make filopodial contacts with TC. Disruption of actin filaments with 100 nM cytochalasin D eliminates filopodia and the frequency of global transients does not increase until growth cones come within 5 mm of the border. n 10 growth cones for each condition; *P < 0.05. (B) Growth cones of spinal neurons plated on LN were scored for crossing onto or turning away from TC in conditions that promote varying frequencies of global and local Ca²⁺ transients. Sixty-seven percent of growth cones turn to remain on LN in 10 mM Ca²⁺ because the frequencies of both global and local transients are higher on TC. More growth cones cross onto TC in 2 mM Ca²⁺ as global and local filopodial Ca²⁺ transient frequencies are reduced. Twenty-five percent of axons turn to remain on LN when global transients are abolished in 0.2 mM Ca²⁺, and the frequency of filopodial transients is not further decreased. Chelating both local filopodial and global growth cone Ca²⁺ transients in 2 mM Ca²⁺ with intracellular BAPTA leads to further crossing from LN onto TC. N 19 growth cones for each condition; *P < 0.01 and **P < 0.005 when compared with 2 mM Ca²⁺, Fisher's exact test.

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Web table 1. Both the incidence and frequency of filopodial Ca²⁺ transients depend on the substrate on which neurons are plated; however, the average duration and amplitude do not. Global growth cone Ca²⁺ transients are associated with filopodial transients on some but not all substrates. N 10 filopodia imaged at 8 Hz for 30 s and n 7 growth cones imaged at .06 Hz for 20 to 30 min for each condition.

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• Movie 1
  Ratio imaging of filopodial Ca²⁺ transients. Thirty-second real-time sequence of intracellular Ca²⁺ dynamics in filopodia detected by ratio imaging of Fluo-4 and Fura-Red fluorescence signals in the region shown in Fig. 1C. Disproportionate changes in Fluo-4/Fura-Red fluorescence (i.e., ratio changes) indicate that these Ca²⁺ transients are not due to filopodial volume changes or movements. Ca²⁺ transients occur in a stationary filopodium, while filopodia that are moving (e.g., to the immediate right of active filopodium) have stable fluorescence ratios. Sequence is comprised of 240 images captured and replayed at 8 Hz. Bar, 2 mm.

• Movie 2
  Propagation of filopodial Ca²⁺ transients. Thirty-second real-time sequence of intracellular Ca²⁺ dynamics in filopodia detected by changes in Fluo-4 fluorescence and presented as a pseudocolored surface plot as seen in Fig. 1E. Note that repeated transient elevations of fluorescence occur independently in several filopodia, but most prominently in the filopodium identified by the white arrow. Some transients in this filopodium initiate near the tip and propagate to the growth cone. Elevations of fluorescence also arise independently in a filopodium crossing the highly active one. Sequence is comprised of 240 images captured and replayed at 8 Hz. Bar, 2 mm.

• Movie 3
  Local Ca²⁺ transients activated by RGDS. Thirty-second real-time sequence of intracellular Ca²⁺ dynamics in a growth cone veil detected by changes in Fluo-4 fluorescence and presented as a pseudocolored surface plot. Images in this movie are rotated 180° from Fig. 2C. RGDS (1 mM) was perfused onto this growth cone during imaging at the point indicated, and after a 14-s refocus period (not shown) this growth cone was imaged in the continued presence of RGDS. Note repeated Ca²⁺ transients locally within the veil on one portion of this growth cone shortly after the addition of RGDS. Bar, 2 mm.