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Figure 4. Organization of sarcomeric proteins actin, myosin, titin, and alpha -actinin in WT versus TEL MO+ myocytes. A-D: Sarcomeric staining for actin, myosin, titin, and alpha -actinin in WT myocytes fixed at 42 hr in culture is highly periodic as compared to immunostained myocytes from TEL MO+ embryos (E-H). Note the distinct titin PEVK doublets visible in WT (C) but not TEL MO+ (G) myocytes. I: Qualitative scoring of WT and TEL MO+ myocytes stained for titin and alpha -actinin; sarcomeric organization of WT cells is significantly better as compared to TEL MO+ myocytes (**P < 0.001). Scale bars = 10 mu m (A, B ,E, F), 2 mu m (C, D, G, H).
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Fig. 2. Telethonin knockdown results in paralysis and kyphosis. A: Bright-field images of uninjected
WT (left), TEL MO� (middle), and SCM (right) stage-42 embryos. WT, TEL MO�, and
SCM embryos are from the same clutch. Note the tail curvature in TEL MO� embryo compared to
WT and SCM embryos. B: Summary data for motility and curvature scores of WT, TEL MO�, and
SCM embryos analyzed at stage 42. Unless stated otherwise, stage-42 embryos are analyzed for
motility and curvature throughout this study. Note the marked decrease in motility and increased
tail curvature in TEL MO� embryos compared to WTand SCM embryos (**P < 0.001).
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Fig. 3. Embryonic and sarcomeric defects in
TEL MO� embryos. Somite and myocyte organization
in WT (A�C, J), TEL MO� (D�F, K),
and SCM-injected embryos (G�I, L). The columns
from left to right are anterior somites (A,
D, G), mid somites (B, E, H), and posterior
somites (C, F, I). Note decreased sarcomere
formation and appearance of wavy and disorganized
myofibrils in TEL MO� somites compared
to WT and SCM. Scale bar � 10 mm.
Primary myocyte cultures obtained from WT
embryos (J) and embryos injected with TEL
MO� (K) or SCM (L). Cell cultures were fixed
at 42 hr after plating and stained for actin
using Alexa-Fluor phalloidin 488. Note the
lack of sarcomeres in TEL MO� myocyte (K)
as compared to highly organized sarcomere
structure in WT (J) or SCM (L). Scale bar � 10
mm. Unless otherwise noted, images in this
and subsequent figures are maximum projections
of Z-series (see Experimental Procedures
section).
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Fig. 5. FL telethonin rescues MO� knockdown both in vivo and in culture. A�C: Somites from
stage-42 embryos co-injected with telethonin mRNA encoding full-length telethonin protein
(MO�FL) show restored sarcomere organization and regular myofibrillar bundling in all regions.
Scale bar � 20 mm. D�F: Primary myocytes in culture from WT-, TEL MO�-, and MO�FLinjected
embryos, fixed at 42 hr after plating. Scale bar � 10 mm.
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Fig. 6. C�del telethonin does not effect rescue of MO� cellular phenotype. A�C: Actin staining
of MO�C�del (C�del: 13 residue truncation at telethonin C-terminal) embryos. Note lack of sarcomeres
and myofibrillar organization in each of the somite regions. Scale bar � 20 mm.
D�F: MO�C�del myocytes fixed at 17, 25, and 42 hr post-plating, respectively. Myocytes have
significantly decreased bundling and sarcomere formation compared to WT (see Fig. 5D) or
MO�FL rescue (see Fig. 6F). Scale � 10 mm.
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Fig. 7. Summary data for MO�FL and MO�C�del rescue experiments. A: Curvature and motility
scores for WT, TEL MO�, MO�FL, and MO�C�del embryos. Note the significant degree of recovery
in morphology and motility after FL rescue compared to TEL MO� and MO�C�del. B: Sarcomere
organization in cell cultures fixed at 17, 25, and 42 hr post-plating. Note that sarcomere
organization is severely disrupted in both TEL MO� and MO�C�del myocytes (**P < 0.001).
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Fig. 8. C-terminal telethonin mutants in MO� background produce defects in bundling and
sarcomere organization. A�C: Images from embryos rescued with C�Ala mRNA in MO� background.
Anterior (A) and mid somites (B) have largely intact sarcomeres but note the wavy
unbundled myofibrils. The posterior somites have fewer sarcomeres and significant actin aggregation
at the somite borders. Scale bar � 20 mm. D�F: In contrast, anterior (D), mid (E), and
posterior (F) somite regions from MO�C�mis (FL residues 1�163, 164 LSRTIVSRIT 173) embryos
have striations but also display actin aggregation and defects in bundling (D�F). Scale bar � 20
mm. G�I, J�L: Myocytes from MO�C�Ala and MO�C�mis fixed at 17, 25, and 42 hr in culture.
G�I: Cultures from MO�C�Ala embryos have few mature sarcomeres and lack myofibrillar organization.
Cells from MO�C�mis fixed at 42 hr (L) have fewer sarcomeres and appear degraded
compared to 17-hr (J) and 25-hr (K) time points. Note the significant difference in striations
between C�Ala and C�mis rescues at the 17- and 25-hr time points. Scale bar � 10 mm.
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Fig. 9. Summary data of MO�C�Ala and MO�C�mis rescue experiments. A: WT, TEL MO�,
MO�C�Ala, and MO�C�mis stage-42 embryos scored for motility and curvature. Note significant
decrease in motility in TEL MO�, MO�C�Ala, and MO�C�mis embryos compared to WT
embryos (**P < 0.001). B,C: Sarcomere organization in MO�C�Ala and MO�C�mis myocytes
fixed at 17, 25, and 42 hr in culture. Note that the decrease in sarcomeric organization in
MO�C�Ala myocytes at each time point is comparable to TEL MO� cells and significantly different
than WT myocytes (B). Sarcomere organization is distinctly decreased in myocytes from
MO�C�mis embryos fixed at 42 hr in culture compared to 17- and 25-hr time points.
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Fig. 10. Myc-telethonin fusion proteins express and localize to the Z-disc. A: Western data for
myc-telethonin overexpression. Lanes 1�5 represent WT, FL, C�del, C�Ala, and C�mis. Note the
expected slight difference in molecular weight for myc-C�del (13 residue truncation, lane 3) compared
to other myc-fusions. B: Localization of myc-telethonin protein at the Z-disk. a�e: Myocytes
from embryos overexpressing myc-telethonin fusion proteins fixed at 42 hr post-plating.
Anti-c-myc staining for each myc-fusion overlaps with the middle of the actin arrays indicating
localization at the Z-disc. Note the diffuse myc staining in WT myocytes (a) compared to myocytes
from embryos injected with the myc-telethonin constructs (rows b�e).
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