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Spencer M
,
Giebelhaus DH
,
Kelly GM
,
Bicknell J
,
Florio SK
,
Milam AH
,
Moon RT
.
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Membrane skeleton protein 4.1 plays a key role in modulating the interactions of spectrin, actin, and integral membrane proteins in erythroid and nonerythroid cells. We have investigated its structure and expression during embryonic development of Xenopus laevis. An analysis of the complete 2758-nucleotide sequence and predicted translation of 801 amino acids (85.5 kDa) of X. laevis oocyte protein 4.1 reveals that, within overlapping regions, oocyte protein 4.1 is 74% identical to a composite amino acid sequence of human erythroid and lymphoid protein 4.1 and has an identity similar to that of amino acid motifs variably expressed in either human erythroid or lymphoid protein 4.1 S1 nuclease protection analysis demonstrates the presence of a single species of protein 4.1 transcript in embryos. Antibodies produced against X. laevis protein 4.1 fusion protein recognize two bands of 180 and 115 kDa on Western blots of X. laevis embryos and retina and, using immunocytochemical techniques, label the developing retina most intensely. In vitro transcription of a cDNA construct fully encoding X. laevis protein 4.1 yields a synthetic mRNA which, when translated in vitro, produces a polypeptide that comigrates on SDS-polyacrylamide gels with the 115-kDa form of embryos and retina. Protein 4.1 is found exclusively in photoreceptors following the terminal mitosis of retinal neurons. When retinal synaptogenesis is complete, protein 4.1 is also expressed in the inner retina. In adult amphibian retinas, protein 4.1 is detected in photoreceptors, bipolar cells, and ganglion cell axons. As these cell types have previously been shown to express spectrin, actin, and ankyrin, it is likely that the membrane skeleton of erythrocytes and retinal cells share functional similarities.
FIG. 1. Comparison of predicted amino acid sequence of X /a+~& oocyte membrane skeleton 4.1 with composite amino acid sequences from
human erythroid and lymphoid protein 4.1 (Tang et al, 1988). Colons (:) denote amino acids which are identical at that position. Motifs, as
previously described in Tang et aL (1988), are denoted with dashed underlining and labeled with human Motifs I and III present in erythroid but
not lymphoid cDNAs and Motif II present in lymphoid but not erythroid cDNAs. The solid underlined sequence is encoded by the cDNA
sequence employed in the Sl nuclease protection assay. The sequence typed in bold, a subset of the underlined sequence, contains the amino
acids used for preparation of the fusion protein and antiserum.
FIG. 3. Immunoblot analysis of erythrocyte membrane proteins,
early X laevis embryos, and retinal proteins using FP02a and chicken
erythrocyte protein 4.1 antisera. Isolated cytoskeletal fractions from
chicken erythroeytes were subjected to two-dimensional gel electrophoresis,
with the direction of isoelectric focusing indicated by the
horizontal arrow and the direction of SDS-gel electrophoresis indicated
by the vertical arrow in A. Identical gels were electrophoretitally
transferred to nitrocellulose and probed with either FP02a antiserum
(A) or chicken protein 4.1 antiserum (B) and localized with
‘%I-labeled protein A. Only the region of the gels with a signal is
shown; the remainder of the blots were unlabeled. Molecular weights
of reactive protein species are noted: 180 kDa (m), 150 kDa (+), 115
kDa (d), 100 kDa (0), and 95 kDa (0). Isolated proteins from embryonic
and adult tissues were then subjected to SDS-gel electrophoresis
and transferred to nitrocellulose (C). Lanes 1, 2, and 3, contain total
protein from 4-, 8-, and 12-day embryos of X Levis, and lanes 4 and 5
contain protein from retina of X laevis and R. pi-, respectively.
Lanes l-5 were probed with the FP02a antiserum. Lanes 6 and 7 are
from a separate experiment. Lane 6 contains protein from retina of R.
@piens which has been blotted to nitrocellulose and probed with
FP02a antiserum as in lane 5, while lane 7 is an adjacent lane on the
blot which contains the [?S]methionine-labeled product of in vitro
translation of the full coding region of X laavis oocyte protein 4.1 (see
Results for details). Bars to the left of the gel indicate molecular
weight markers of 200,116,97, and 66 kDa.
FIG.~. II nmunolabeling of protein 4.1 in developing and adult X laevis retina with affinity-purified FP02a antibodies. (a, c, e) I Sections from the central retinas of 4-, 5-, and 8-day embryos were stained with Richa .rdson’s and viewed with transmitted light. Fluorescence I micrographs of
protein 4 .l labeling of the same retinas are shown in b, d, and f. (a, 1: I) Four-day (stage 32) embryo. (a) The neuroblastic retina apposes the
retinal pigment epithelium (rpe) and is not stratified, and plexiform layers cannot be discerned. (b) No fluorescent signal can be detected with
protein 4.1 antiserum. (c, d) Five-day (stage 37) embryo. (c) The photoreceptors have rudimentary outer segments (arrowhead) and outer (small
asterisks) and inner (large asterisk) plexiform layers which separate the nuclear layers (onl, outer nuclear layer; inl, inner nuclear layer; gel,
ganglion cell layer). Ganglion cell axons form the optic nerve (on) which exits the retina. 1, lens. (d) Fluorescent label is localized in the
developing photoreceptors (pr) distal to their nuclei. (e, f) Eight-day (stage 49) embryo. (e) The retina appears adult-like except that
photoreceptor outer segments (small arrowhead) have not fully elongated. Cones can be discerned from rods by the presence of an oil droplet in
the inner segment (large arrowhead). The inner plexiform layer (large asterisk) has expanded in width. (f) Photoreceptor outer (small
arrowhead) and inner segments and the outermost somata of the inner nuclear layer label. No fluorescence can be seen in the cone oil droplets
(large arrowhead) or the outer nuclear layer. Diffuse label is seen in the inner plexiform (large asterisk) and ganglion cell layers. (a-f) Bar, 20
pm.
FIG. 5. Immunolabeling of protein 4.1 in adult R. pipiens retina wit
affinity-purified FP02a immune (a, b) and preimmune antibodies (c
(a) Entire bipolar cells label from their dendritic terminals in the outer plexiform layer (small asterisks) to their axonal terminals (arrowhead)
in the inner plexiform layer (large asterisk). Photoreceptors
(OS, outer segments; is, inner segments; onl, outer nuclear layer)
and ganglion cell somata (gel, ganglion cell layer) do not label. (b)
Ganglion cell axons, which form the optic nerve (on) label. (c) No label
is seen with FP02a preimmune serum. rpe, retinal pigment epithebum.
(a-c) Bar, 20 pm.
