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Cell adhesion is crucial for migration of cells during development, and cell-substrate adhesion of motile cells is accomplished through the formation and removal of focal complexes that are sites of cell-substrate contact. Because Ca2+ signaling regulates the rate of axon outgrowth and growth cone turning, we investigated the potential role of Ca2+ in focal complex dynamics. We describe a novel class of localized, spontaneous transient elevations of cytosolic Ca2+ observed both in Xenopus neuronal growth cones and fibroblasts that are 2-6 mum in spatial extent and 2-4 s in duration. They are distributed throughout growth cone lamellipodia and at the periphery of fibroblast pseudopodia, which are regions of high motility. In both cell types, these Ca2+ transients lead to disappearance of phosphorylated focal adhesion kinase (pFAK) and deadhesion from the substrate as assessed by confocal and internal reflection microscopy, respectively. The loss of pFAK is inhibited by cyclosporin A, suggesting that these Ca2+ transients exert their effects via calcineurin. These results identify an intrinsic mechanism for local cell detachment that may be modulated by agents that regulate motility.
Fig. 3. Regulation of pFAK immunofluorescence by integrin activation. (A) pFAK staining in control (2 mM Ca2+ medium). (B) +RGDS (1 mM). (C) +RGDS + 10
mM Ni2+. (D) + RGES (1 mM). Neurons were grown on tissue culture plastic. Images are inverted (immunofluorescence is black). (E) The mean pFAK staining
intensity is significantly increased in cells incubated with the integrin activating peptide RGDS for 5 min prior to fixation. This effect is not blocked by the prior
addition of Ni2+ to prevent LLTs, nor is it achieved by the non-integrin activating peptide RGES. n 10 growth cones for each condition. Asterisks denote
statistically different from 2 mM Ca2+ (p < 0.05) and not different from each other.
Fig. 4. pFAK staining is influenced by factors that reduce LLT frequency. (A) Growth cones of cells grown in 0 mM Ca2+ are more densely stained than those of cells
grown in 2 mM Ca2+ (Fig. 3A). (B, C) Light staining in the presence of osmotically balanced 30 mM extracellular Ca2+ is converted to dense staining by exposure to
100 nM BAPTA-AM. (D) The mean pFAK staining intensity is decreased significantly when cells are grown in the presence of extracellular Ca2+; this effect is blocked
by addition of BAPTA to suppress elevation of intracellular Ca2+ (Gu and Spitzer, 1995). n > 20 growth cones for each condition. Asterisk denotes statistically
different from 0 mMCa2+ and 30 mM Ca2+ plus BAPTA (p < 0.05). (E) pFAK staining is more intense in cells grown in 2 mMCa2+ on tenascin where LLTs are absent
than on tissue culture plastic where LLTs are present; arrows identify two growth cones on these different substrates in the same culture, �200 Am apart.
Fig. 5. pFAK immunoreactivity colocalization with focal complexes in lamellipodia. (A, B) Images of the growth cone of a cultured neuron fixed and stained for
pFAK-IR (red) and examined using IRM (green). Inset in panel A shows growth cone region of interest. Merged images at intermediate (C) and high magnification
(D, boxed region in panel C) reveal that most of the red spots representing focal complexes are in register with green spots (yielding yellow), indicating close cell �
substrate contact in the distal regions of the veil of the lamellipodium (examples indicated by white arrowheads).
Fig. 7. The site of LLT generation coincides with low pFAK immunoreactivity.
(A) Fluo-4 fluorescence image of a growth cone generating an LLT (arrow). (B)
pFAK immunoreactivity of the same cell 5 min following the incidence of the
LLT shows reduced staining in the region of the growth cone where the LLT
occurred (yellow dashed circle). This result was observed in 6 of 6 neuronal
growth cones that were fixed and stained for pFAK following the observation
of an LLT during Ca2+ imaging. (C) Fluo-4 image of a growth cone not
generating an LLT during the period of imaging. (D) There are no regions of
distinctively low intensity pFAK immunoreactivity in the growth cone. Results
similar to panels C and D were obtained from 6 of 6 growth cones.
Fig. 9. The site of LFT generation coincides with low adhesion and low pFAK
immunoreactivity. (A) Surface plot of fluo-4 fluorescence in three fibroblasts,
one of which generated a localized transient during the period of imaging
(arrow). (B) The culture was fixed 5 min after imaging the LFT, and the same
fibroblasts were analyzed by IRM. The location of the LFT (yellow dashed
circle) coincides with brighter intensity in the IRM image. Similar results were
obtained from 5 of 5 cells. (C) Surface plot of fluo-4 fluorescence in another
fibroblast, which generated an LFT. (D) The culture was fixed 5 min after
imaging the LFT and stained for pFAK. The site of LFT generation corresponds
to a localized reduction in pFAK immunoreactivity. Results similar to panels C
and D were obtained from 7 of 7 growth cones.
Fig. 10. Cyclosporin A suppresses the local reduction of pFAK immunoreactivity
by LFTs. (A) Surface plot of fluo-4 fluorescence in two fibroblasts in the
presence of 10 nM cyclosporin A, each of which generated a localized transient
during the period of imaging (arrows). (B) The culture was fixed 5 min after
imaging, and the same fibroblasts were stained for pFAK. There was no
detectable reduction in pFAK immunoreactivity at the site of LFT generation
compared to non-LFT generating sites in the same cell. The locations of the
LFTs are indicated by yellow dashed circles. Similar results were obtained from
5 of 5 cells. (C) Surface plot of fluo-4 fluorescence in another fibroblast in the
absence of cyclosporin A (control), which generated a LFT (arrow). (D) The
culture was fixed 5 min after imaging the LFT and stained for pFAK. The site
of LFT generation corresponds to a localized reduction in pFAK immunoreactivity.
Labeling of cultured spinal neurons by Rabbit anti- phosphorylated-Ptk2 antibody
Ptk2 (protein tyrosine kinase 2) gene expression in spinal neurons cultured from Xenopus laevis embryos, NF stage 15, as assayed by immunofluorescence.
