doi: 10.1242/10.1242/dev.00545
Interactions between the secreted protein Amalgam, its transmembrane receptor Neurotactin and the Abelson tyrosine kinase affect axon pathfinding
Eric C. Liebl1,
,
R. Grant Rowe1,
David J. Forsthoefel2,
Amanda L. Stammler1,
Erica R. Schmidt1,*,
Michelle Turski2,
and
Mark A. Seeger2
1 Department of Biology, Denison University, Granville, OH 43023, USA
2 Department of Molecular Genetics and The Center for Molecular Neurobiology,
The Ohio State University, Columbus, OH 43210, USA
* Present address: College of Medicine, The Ohio State University, Columbus, OH
43210, USA
Present address: University Program in Genetics, Duke University, Durham, NC
27708, USA
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Fig. 2. Biochemical characterization of Ama+ and AmaM109. (A)
The organization of Ama with its signal sequence and three Ig domains (loops).
The N terminus is towards the left. The amaM109 and the
amaR1 mutations are also indicated. (B) Representative
photomicrographs showing our S2 cell adhesion assay at t=8 hours. In
the top three panels, all cells were engineered to express wild-type Nrt and
were exposed to either control (left), Ama+-containing (center) or
AmaM109-containing (right) conditioned media. In the bottom two
panels, cells were engineered to express membrane-anchored forms of Ama. (C)
Quantification of our S2 cell adhesion assays. Bars represent the percent of
total particles at t=0 hours counted at t=8 hours. Values
represent the average of two independent experiments; error bars show the
standard error of the mean. (D) Anti-Ama immunoblots. The arrow indicates the
mobility of full-length Ama. Lanes 1, 2: equal amounts of
Ama+-conditioned media or of AmaM109-conditioned media
were resolved. These lanes show that comparable amounts of Ama were present in
both types of conditioned media. Lanes 3-6: immunoblots of S2 cell lysates
from cell pull-down assays. Equivalent amounts of cell lysates were loaded in
each lane. Lane 3: naïve S2 cells exposed to Ama+-conditioned
media. Lane 4: Nrt-expressing S2 cells exposed to Ama+-conditioned
media. Lane 5: naïve S2 cells exposed to AmaM109-conditioned
media. Lane 6: Nrt-expressing S2 cells exposed to
AmaM109-conditioned media. These lanes show that Ama+
and AmaM109 bound specifically to S2 cells expressing Nrt. (E)
Anti-Nrg immunoblots. Equivalent amounts of cell lysates were loaded in each
lane. The arrow indicates the mobility of the Ama-Nrg fusion protein. Lane 1:
S2 cells expressing Ama+-TM. Lane 2: S2 cells expressing
AmaM109-TM. These lanes show equivalent amounts of these chimeric
proteins were expressed.
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Fig. 1. CNS phenotypes of Abl and ama mutants. Embryos from stage
14/15 were stained with mAb BP102 and CNSs were dissected. Representative
examples of the overall phenotype associated with each genotype are shown. (A)
Abl1/Abl4, (B) Abl1,
amaM109/Abl4, (C) Abl1,
Df(3R)ama/Abl4, (D) Abl1,
amaR1/Abl4, (E) Abl1,
amaR1/Abl4, Df(3R)ama, (F)
Abl1, amaM109/Abl4,
Df(3R)ama. Percentages below each panel indicate the
percent of segments with commissure defects; n=total segments scored.
Arrows in B,E show typical examples of segments scored as having defective
commissures.
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Fig. 3. CNS phenotypes of Abl and nrt mutants. Mutants were
either generated by RNAi (A-C) or by zygotic genotypes (D-F). Embryos from
stage 14/15 were stained with mAb BP102, and CNSs were dissected.
Representative examples of the overall phenotype associated with each genotype
are shown. (A) Abl null, (B) nrt null, (C) Abl and
nrt null, (D) Abl1,
nrtM54/Abl4, (E) Abl1,
nrtM100/Abl4, (F)
Df(3L)st-j7, nrtM2/Abl1,
nrtM54. Percentages below each panel indicate the percent of
segments with commissure defects; n=total segments scored. Arrows in
D,E show typical examples of segments scored as having defective
commissures.
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Fig. 4. Biochemical characterization of NrtM100 and NrtM221.
(A) The Nrt protein is represented. The N terminus is towards the left. The
location of mutations nrtM29, nrtM221,
nrtM54, nrtM2 and
nrtM100 are also shown. nrtM54 is an
11 bp deletion that results in a frameshift (fs). (B) Graphical representation
of the quantification of our S2 cell adhesion assay. Bars represent the
percent of total particles at t=0 hours counted at t=8
hours. Values represent the average of two independent experiments; error bars
show the standard error of the mean. (C) Immunoblots of S2 cell lysates from
cell pull-down assays. (Top) Anti-Nrt immunoblots. Equivalent amounts of cell
lysates were loaded in each lane. The arrow shows the migration of full-length
Nrt. Lane 1: naïve S2 cells. Lane 2: S2 cells engineered to express
Nrt+. Lane 3: S2 cells engineered to express NrtM100.
Lane 4: S2 cells engineered to express NrtM221. Naïve S2 cells
did not express Nrt, and all Nrt proteins were expressed from the pMET plasmid
at comparable levels. (Bottom) Anti-Ama immunoblots. Equivalent amounts of
cell lysates were loaded in each lane. The arrow shows the migration of
full-length Ama. Lane 1: naïve S2 cells. Lane 2:
Nrt+-expressing S2 cells. Lane 3: NrtM100-expressing S2
cells. Lane 4: NrtM221-expressing S2 cells. Only S2 cells
expressing wild-type Nrt bound Ama.
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© The Company of Biologists Ltd 2003
