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Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the regulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane specialization. In this study, we examined the immunocytochemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sarcolemmal specializations, the myotendinous junction and the neuromuscular junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demonstrated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phosphotyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its distribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like specializations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal specializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetylcholine receptor clusters formed in culture, it does not appear to be involved in the development of the neuromuscular junction.
Fig. 1. FAK in Xenopus embryos
detected by immunoprecipitation with
mAb 2A7 and immunoblotting with
polyclonal antibody BC3. Lane A,
sample containing embryonic lysate;
lane B, sample without lysate. A
specific band at 116 kDa (arrowhead)
was detected in the lysate sample and it
was absent from the lysate-free sample.
Other bands present in both lanes are
non-specific and include heavy and light
chains of the IgG molecules used in the
reaction.
Fig. 2. FAK immunolabeling at the MTJ in dissociated Xenopus
tadpole myotomal muscle fibers. Tadpole tails were skinned, and
myotomal fibers were dissociated in collagenase. (A) Phase-contrast
image of the tip of one of these multinucleated fibers. Arrowhead
points to the position of a NMJ. Several satellite cells can be
observed in close association with the sarcolemma. (B) FITC-FAK
immunolabeling showing the distribution of FAK along the extent of
the MTJ at the fiber tip. MTJ infoldings are shown by the arrows.
(C) R-BTX-labeling of the NMJ (arrowhead). Comparison of the
FAK labeling with the NMJ (B-C) shows that the this protein is
absent from the postsynaptic AChR clusters.
Fig. 3. PY immunolabeling in dissociated Xenopus tadpole myotomal muscle fibers. Tadpole tails were skinned, and myotomal fibers were
dissociated in collagenase. All procedures after fixation were carried out at 4°C, and all solutions contained 1 mM Na3VO4. Two focal planes
(top and bottom rows) of the same cell are presented. Left column (A and C), FITC-PY. Right column (B and D), R-BTX. Note the
concentration of FITC-PY labeling along the extent of the MTJ (filled arrows, A), but also the colocalization of PY at the NMJ (compare open
arrowheads in A and B, and filled arrows in C and D).
Fig. 4. FAK immunolabeling in adult Xenopus leg muscle. Cryosections (8 mm) were labeled with R-BTX and subjected to the immunolabeling
protocol. (A-E) FITC-FAK. (F) R-BTX. (A) FAK labeling caps the tips of muscle fibers (filled arrows) as they insert into the tendon at the
MTJ. Labeling decreases dramatically along the edge of the fibers moving away from the junctional area (open arrowheads). (B) A higher
magnification image of a single fiber, showing the MTJ infoldings labeled with FAK at the tip, labeling along the proximal edge of the fiber,
and the decrease in staining further away from the tip. (C) Control in which the primary 2A7 anti-FAK mAb was preincubated with lysate of
SF21 cells expressing FAK cDNA. (D) A low level of FAK immunolabeling (open arrowhead) is observed along the sarcolemma at
extrajunctional regions of the muscle. (E-F) The same low level of FAK labeling along the sarcolemma (E, filled arrows) is observed at the
NMJ (F), but no concentration of FAK is observed. The bright structures in (E-F) are most likely autofluorescence due to satellite cells.
Fig. 5. Concentration of FAK immunolabeling at AChR hot spots. Embryonic Xenopus muscle cells were plated and maintained in culture for
several days. Left column, R-BTX. Right column, FITC-FAK. (A-B) Several AChR hot spots show colocalized FAK labeling. Several
examples are shown by the arrows. This high level of colocalization was the pattern most often observed. Less often, AChR clusters did not
demonstrate coextensive FAK accumulation. (C-D) Three AChR hot spots have formed (arrowheads), but only two show FAK labeling.
(E-F) Two hot spots have formed (arrowheads), but neither shows an accumulation of FAK labeling.
Fig. 6. Control of FAK immunolabeling at AChR
hot spots in cultured muscle cells. Left column,
R-BTX. Right column, FITC-FAK. (A-B) 2A7
was preabsorbed with 20-fold excess recombinant
FAK expressed by transfected SF21 cells. The
FAK labeling at AChR clusters was abolished.
(C-D) Preincubation of mAb 2A7 with lysate
from non-transfected SF21 cells had no effect on
FAK labeling at AChR clusters.
Fig. 7. No FAK immunolabeling is detected at NMJs formed in
culture. (A) Phase-contrast image of a neurite-muscle cell contact
(arrows). (B) R-BTX labeling of an AChR cluster induced to form
along the extent of the nerve-muscle contact, indicative of a
functional NMJ. (C) FITC-FAK immunolabeling showing the lack of
FAK accumulation at the NMJ.
Fig. 8. Concentration of FAK immunolabeling at AChR clusters
induced by polystyrene beads; 10 mm beads were applied to muscle
cells and cultures were incubated for 24 hours. Left column, R-BTX.
Right column, FITC-FAK. (A-B) Four bead contacts induced AChR
clusters and colocalized FAK labeling (arrows). (C-D) A single bead
contact induced AChR clustering with colocalized FAK, as well as
an accumulation of FAK at a site lacking AChRs (arrow). (E-F) A
single bead contact induced clustering at two distinct sites on two
muscle cells (arrows), but only one of these sites demonstrates FAK
accumulation. (G-H) Three beads induced FAK accumulation
(arrows), but only two of these sites show clustered AChRs.
Fig. 9. Time course for the development of FAK immunolabeling
relative to AChR cluster formation at bead contacts; 10 mm beads
were applied to cells and allowed to incubate for various times. By
16 hours the percentage of beads that induced AChR clusters had
plateaued at 80%, whereas only 30% of contacts demonstrated FAK
labeling. By 24 hours, 50% of contacts showed FAK accumulation,
and by 48-72 hours, almost all of the bead contacts that induced
AChR clustering also induced FAK accumulation. 160 cells were
analyzed per time point over two experiments. Values are mean ±
s.e.m.
Fig. 10. Fluorescence images of the development of FAK immunolabeling relative to AChR cluster formation at bead contacts; 10 mm beads
were applied to cells and allowed to incubate for various times. Left column, R-BTX. Middle column, FITC-FAK. Right column: phasecontrast.
(A-C) By 16 hours, AChR clusters are well formed, but the relatively small number of contacts that induce FAK accumulation
demonstrate a light, punctate FAK pattern. (D-F) By 48 hours, the intensity of FAK labeling increased, and the pattern more closely
approximated the pattern of clustered AChRs. (G-I) By 72 hours, the intensity of FAK labeling increased even more, and appeared to be
brighter than the AChR signal at many contacts.
