Mansi R Khanna, Floyd J Mattie, Kristen C Crilly,

Cell Biology:
Spectrin Tetramer Formation is not
Required for Viable Development in
Drosophila
Mansi R Khanna, Floyd J Mattie, Kristen C
Browder, Megan D Radyk, Stephanie E Crilly,
Katelyn J Bakerink, Sandra L Harper, David
W Speicher and Graham H Thomas
J. Biol. Chem. published online November 7, 2014
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2014/11/07/M114.615427.DC1.html
This article cites 0 references, 0 of which can be accessed free at
http://www.jbc.org/content/early/2014/11/07/jbc.M114.615427.full.html#ref-list-1
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
Access the most updated version of this article at doi: 10.1074/jbc.M114.615427
JBC Papers in Press. Published on November 7, 2014 as Manuscript M114.615427
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.615427
Drosophila development without spectrin tetramerization
Spectrin tetramer formation is not required for viable development in Drosophila*
Mansi R. Khanna1,3, Floyd J. Mattie1, Kristen C. Browder1, Megan D. Radyk1, Stephanie E. Crilly1,
Katelyn J. Bakerink1, Sandra L. Harper2, David W. Speicher2 and Graham H. Thomas1,4
1
The Department of Biology and The Department of Biochemistry and Molecular Biology
The Pennsylvania State University, University Park, PA, 16802
2
3
Systems Biology Division, The Wistar Institute, Philadelphia, PA, 19104
current address: Center For Neurodegenerative Disease Research, University of Pennsylvania,
Philadelphia PA 19104
*Running title: Drosophila development without spectrin tetramerization
Keywords: Drosophila, cytoskeleton, cell biology, protein assembly, membrane proteins,
spectrin, membrane skeleton
α -specR22S,
a
spectrins.
However,
tetramerization site mutant homologous to the
pathological α -specR28S allele in humans,
eliminates detectable binding to β-spectrin and
reduces binding to β H-spectrin ~1000 fold. Even
though spectrins are essential proteins, α specR22S rescues α-spectrin mutants to
adulthood with only minor phenotypes
indicating that tetramerization, and thus
conventional network formation, is not the
essential function of non-erythroid spectrin.
Our data provide the first rigorous test for the
general requirement for tetramer-based nonerythroid spectrin networks throughout an
organism and find that they have very limited
roles, in direct contrast to the current
paradigm.
Background: Underneath the membrane of most
animal cells is a network (membrane skeleton)
assembled using tetramers of the protein spectrin.
Results: While spectrins are essential proteins,
tetramer formation is surprisingly unimportant for
Drosophila development.
Conclusion: The major roles of the membrane
skeleton do not require a conventional network.
Significance: The ubiquitous model of the
spectrin based membrane skeleton has limited
applicability.
ABSTRACT
The dominant paradigm for spectrin
function is that (αβ)2-spectrin tetramers or
higher order oligomers form membrane
associated two-dimensional networks in
association with F-actin to reinforce the plasma
membrane. Tetramerization is an essential
event in such structures. We characterize the
tetramerization interaction between α-spectrin
and β-spectrins in Drosophila. Wild-type αspectrin binds to both β- and β H-chains with
high affinity, resembling other non-erythroid
α− and β-Spectrins form (αβ)2 heterotetramers
to crosslink F-actin and form spectrin based
membrane skeletons (SBMS)5. Analysis of the
erythrocyte has provided the dominant paradigm
for the SBMS, where short F-actin rods are
interconnected by spectrin to form a branching
1
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
To whom correspondence should be addressed: Graham H Thomas, Department of Biology, Department
of Biochemistry and Molecular Biology, 208 Mueller Laboratory, The Pennsylvania State University,
University Park, PA 16802, USA, Tel.: (814) 863-0716; email; Fax: (814) 865-9131; E-mail:
[email protected]
Drosophila development without spectrin tetramerization
EXPERIMENTAL PROCEDURES
Constructs
and
Spectrin
Fragment
Purification - Spectrin gene fragments encoding
the tetramerization domain were amplified (Table
1) from preexisting cDNA clones (α-spectrin, βspectrin, βH-spectrin) or mutated constructs (αspecR22S), cloned into pGEX4T-1, sequence
verified and expressed in E. coli BL21-CodonPlus
(DE3)-RIPL (Agilent Technologies, Santa Clara,
CA) using standard methods. Bacteria were grown
to log phase in LB and induced using 1mM IPTG.
Fusion proteins were extracted in Lysis Buffer
(50mM Tris.Cl pH8.0, 50mM NaCl, 1mM βmercaptoethanol [βME], 5mM EDTA, 150µM
PMSF, 1µg/ml Leupeptin, 1 µg /ml Pepstatin,
1 µg/ml DFP) by sonication on ice. After
centrifugation the supernatant was mixed with
glutathione-agarose beads (Sigma-Aldrich, St.
Louis, MO) that had been previously equilibrated
in PBEP (10 mM NaPO4, 130 mM NaCl pH7.3,
1mM βME, 5mM EDTA and 150µM PMSF).
After 1hr of tumbling, the beads were packed into
a column and washed with ≥10 bed volumes of
PBEP, followed by ≥10 bed volumes of PBEP
without PMSF, and eluted using G-buffer (50mM
Tris.Cl pH8.0, 10mM reduced Glutathione, 1mM
2
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
proteins (20-23), but data on the general
requirement for oligmerization via the H2H
interaction per se, and therefore conventional
network formation dependent upon F-actin
crosslinking, is lacking.
The analysis of the temperature sensitive (Ts)
α-specR22S mutation appeared to indicate that
network formation is essential for viability in
Drosophila (24). We were interested in utilizing
the reported Ts network formation and
quantitatively characterized the biochemical
consequences of this mutation. We find α-specR22S
dramatically affects H2H binding at all
temperatures, but that full-length α-spectrinR22S
rescues adult development in a null background.
Reexamination of the phenotypic consequences of
this mutation reveals only minor defects in the
ovaries, midgut and at neuromuscular junctions,
and that the previously attributed phenotype is
actually due to poor fly husbandry. We conclude
that tetramerization of α/β-spectrin has very
limited roles in fly development, in direct contrast
to the red cell paradigm.
network resembling a fishing net (1,2). αI/βIspectrin will also form higher order oligomers
(heterotetramers, hexamer and octamers etc.) (3)
and recent cryoelectron microscopy images have
indicated that these are common in the red cell (4).
Spectrin oligomerization via the head-to-head
(H2H) interaction between the N-terminus of αIspectrin and a C-terminal domain of βI-spectrin is
required to form this network. In the erythrocyte,
the H2H interaction is of moderate affinity
(Kd=400-800nm) (5,6) permitting network
formation, but also rearrangement upon shear
stress during circulation (7). Mutations that
decrease H2H affinity reduce or eliminate
oligomerization and cause network disruption and
hereditary
elliptocytosis
or
hereditary
pyropoikilocytosis (e.g. 6,8,9), so it is clear that
the demands for such a network are acute in red
cells.
H2H binding between vertebrate nonerythroid αΙΙ- and βΙΙ-spectrins is much tighter
(Kd=4.5-8.5nm) (5), but the morphology of the
non-erythroid SBMS is less well defined. F-actin
crosslinking by spectrin has been observed in vitro
(10), in the terminal web (11), on the membrane
of inner hair cells (12) and in some axons (13). In
these cases the models proposed are all based on
oligomeric states that form via the H2H
interaction, an interaction that has apparently
persisted for some 500 Myr (see 14).
In most cell types the exact form of the SBMS
remains undefined (e.g. 15,16), and other network
organizations
may
exist:
Alternative
oligomerization mechanisms based on lateral
interactions between α-spectrin and β-spectrin
have been proposed (17) but remain equivocal
(18). Recently, it was shown that human βHspectrin can self associate (19), raising the
possibility that F-actin crosslinking could be
mediated by this β isoform in the absence of αspectrin. Clearly there is potential for diversity
although none has yet been demonstrated. For the
purposes of this paper we refer to any H2Hdependent oligomerization process that leads to Factin crosslinking as the ‘conventional network’ –
this being the activity upon which all currently
visualized networks appear to depend.
Genetic investigations have demonstrated that
non-erythroid α- and β-spectrins are essential
Drosophila development without spectrin tetramerization
3
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
calorimeters (GE Healthcare LifeSciences,
Piscataway, NJ). Samples were dialyzed overnight
in 10 mM Na PO4, 130 mM NaCl, 1 mM βmercaptoethanol, pH 7.4 before analysis, and
wild-type β-spectrin 16-17 at or wild-type βHspectrin 31-32 at 10-15 mM or dialysate buffer
(controls) were loaded into the reaction cell. Wildtype or mutant α-spectrin 0-1 at a concentration of
95-185 mM were used as the titrant for
experimental measurements and controls at 18°C,
23°C or 29°C stirring at 300 rpm in the reaction
cell. Typically, 26 injections of titrant (10 µL each
with VP-ITC; 1.5-2µl each with Auto iTC200).
Protein concentrations were determined from
absorbance at 280nm. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was used to
verify the absence of degradation products before
and after each experiment. Data were analyzed
using OriginLab (v5.0) software as previously
described (25,26) to calculate dissociation
constants. All interactions were characterized at
least three times.
Fly strains - The transformation host strain y
w was used as a wild-type control. The wild-type
UAS-myc-α-spectrin37 strain (27) and lines
containing α-speclm32 α-speclm51 and α-speclm111
alleles were a gift from Dr. Ronald Dubreuil
(University of Illinois, Chicago, IL). Lines
containing α-specrg41 and Df(3L)RR2 were a gift
from Dr. Daniel Branton (Harvard University,
Cambridge, MA).
All of the α-spec alleles were originally
generated on a roughoid scarlet ebony
chromosome (28); as a result lm/lm or lm/rg allelic
combinations exhibit these phenotypes. α−specrg41
is a null allele that is known to be a deletion that
results in a frameshift after just 91 residues
(20,29,30). α-speclm32 and α-speclm111 have not
been sequenced but do not produce detectable
protein and are either null or strong hypomorphs
(20,29,30). α-speclm51 is lethal over Df(3L)RR2and
other α-spec alleles, but is otherwise
uncharacterized (20,29,30).
The germline MTD-Gal4 driver was obtained
from the Bloomington stock center (BL #31777;
Indiana University, Bloomington, IN). The αspectrin GFP line contains GFP inserted between
amino acids 1330/1 (repeat 12) at the endogenous
locus after a weeP GFP-exon-trap insertion (31).
βME, 5mM EDTA, 1 µg/ml Pepstatin). Thrombin
(Sigma-Aldrich, St. Louis, MO) was dissolved in
dH2O, added at an empirically determined ratio to
release GST from each spectrin fragment and
stopped by the addition of 300µM PMSF.
Following extensive dialysis in PBEP the
GST/spectrin mixture was again applied to
glutathione beads as described above to remove
the GST. Flow through from this column was
concentrated and residual contaminants were
removed by passing it through a preparative
HiLoad 16/60 Superdex 75 column. MALDI
analysis of each protein verified that the mass of
all fragments was as expected (Table 1). Circular
dichroism and analytical ultracentrifugation were
performed as previously described (6) to verify
that each protein had a prototypical helical content
for such spectrin fragments and that they behaved
as monomers. Proteins were stored on ice until
used.
HPLC analysis of complex formation –
Complex formation between α-spectrin and βspectrin fragments was verified by gel filtration.
~1nmol of each fragment alone or in combination
was combined, allowed to assemble on ice
(assembly was complete in <1min), then loaded
directly on to two analytical (7.8 x 300 mm) TSK
gel columns (G3000SWXL and G2000SWXL) in
series (Tosoh, King of Prussia, PA) equilibrated in
10 mM Na PO4, 130 mM NaCl, 1 mM EDTA,
0.15 mM PMSF, 1 mM β-mercaptoethanol pH 7.3.
Differential scanning calorimetry - Thermal
denaturation analyses of spectrin recombinant
peptides were performed using an MCS
differential scanning calorimeter (MicroCal,
Northampton, MA). Purified proteins were
exhaustively dialyzed against 10 mM Na PO4, 130
mM NaCl, 1 mM β-mercaptoethanol, pH 7.4.
Samples were degassed for 5 minutes, and protein
samples at 0.6 to 1.0 mg/mL in the sample cell
were compared with dialysate buffer in the
reference cell by scanning from 25°C to 100°C at
a scan rate of 90°C/h. Data analysis was
performed using the ORIGIN (v3.2) software
provided with the instrument.
Isothermal
titration
calorimetry
Thermodynamic parameters for the binding of the
α-spectrin 0-1 recombinant fragments to wild-type
16-17 were determined using a MicroCal VP-ITC
or MicroCal Auto-iTC200 isothermal titration
Drosophila development without spectrin tetramerization
4
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
supernatant 3A9 (1:1,000-10,000 / 1:20; from The
Developmental Studies Hybridoma Bank); mouse
anti-c-myc 9E10 (1:100 / 1:10; Enzo Life
Sciences, Farmingdale, NY); Alexafluor 488conjugated goat anti-Horseradish Peroxidase
(1:500; Jackson ImmunoResearch); Rabbit antiSynaptotagmin Dsyt-CL1 (1:5000; Dr. Noreen
Reist, Colorado State University, Fort Collins,
CO), mouse anti-Hts (Adducin, 1B1; 1:25)
developed by Dr. Howard Lipshitz and mouse
anti-Discs large (4F3; 1:50) developed by Dr.
Corey Goodman were obtained from The
Developmental
Studies
Hybridoma
Bank
developed under the auspices of the NICHD and
maintained by The University of Iowa,
Department of Biology, Iowa City, IA 52242.
HRP-conjugated anti-mouse secondary antibodies
for immunoblots (Jackson Immunoresearch, West
Grove, PA) were used at 1:2500. Alexafluor-405, 488 and -594 labeled goat secondary antibodies
(Invitrogen, Carlsbad, CA) were preadsorbed
against fixed embryos and used at 1:250.
Immunoblots
used
enhanced
chemiluminescent
substrate
(Pierce/Thermo
Fisher, Rockford, Il) and were quantified either
using a blot scanner (Licor, Lincoln, NE) or with
appropriate film exposures (that utilized serial
dilutions to verify linear response of the
emulsion), followed by imaging and densitometry
in Image J (imagej.nih.gov).
Immunostaining of ovaries was performed as
previously described (33). To visualize F-actin
staining in the brush border, guts from wandering
third-instar larvae midguts were dissected and
accumulated on ice in PBS (130mM NaCl, 10mM
Na PO4, pH7.4), then fixed for 20 minutes in 8%
formaldehyde in PBS at room temperature.
Phalloidin staining was performed as previously
described (34).
For immunocytochemistry of neuromuscular
junctions (NMJ), wandering third instar larvae
were dissected leaving the CNS in place and
pinned out to expose the body wall muscles,
immediately fixed in 4% formaldehyde in PBS for
20 minutes, rinsed with PBS, and the CNS
removed and discarded. The remaining body wall
muscle “pelt” preparation was unpinned and
further processed for immunocytochemistry in
glass depression dishes. PBS containing 0.1%
Triton X-100 and 5% normal goat serum was used
for subsequent antibody incubations and washes.
This strain is homozygous viable, exhibits wildtype α-spectrin distributions and was a gift from
Dr. Melissa Rolls (Penn State, University Park,
PA).
All available α-specR22S fly lines no longer
expressed α-spectrin (see text). Since, we sought
to achieve the same Ts rescue of α-spectrin null
mutations shown by Deng et al. we exactly
replicated their construct (32). Plasmid
P[w+UPS] containing a full-length wild-type αspectrin cDNA (Lee et al., 1993; a gift from Dr.
Ronald Dubrueil, University of Illinois, Chicago,
IL) was mutated using the QuikChange∆ II XL
mutagenesis kit (Agilent Technologies) to create
the R22S substitution. In addition, we similarly
transformed an NcoI site at the start codon to KpnI
to permit cloning into P[w+UM-2], a P-element
vector containing the constitutive ubiquitin
promoter and Myc tag as previously described
(24,32). The entire gene was sequenced after each
round of mutagenesis and in the final construct to
ensure
there
were
no
non-synonymous
substitutions. Rainbow Transgenic Flies, Inc.
(Camarillo, CA) created fly strains according to
standard methods using a y w host. As soon as
possible all inserts were combined with a copy of
the α-specrg41 null allele to make stocks that
should be less susceptible to the effects of chronic
α-spectrin overexpression.
To create α-specR22S rescued flies the crossing
scheme outlined in Figure 3 was followed. With
these two crosses rescued flies containing 1, 2 or 3
copies of the α-specR22S transgene were generated
and designated 1, 2 or 3 copy rescue (1cr, 2cr, 3cr)
respectively. In addition, siblings that contained 1,
2 or 3 copies of the α-specR22S transgene in the
presence of one wild-type allele of α-spectrin were
generated and designated 1, 2 and three copy
coexpression (1cx, 2cx, 3cx) respectively. Rescue
to adulthood was quantified as a percentage of the
one copy coexpression class in crosses producing
an average of 490 offspring (range: 277-721) and
included only eclosed and active flies.
Antibodies and imaging - The following
primary antibodies were used at the dilutions
indicated in parentheses (immunoblot /
immunofluorescence): one of two preparations of
mouse anti-α-Spectrin were used: Ascites #N3
(1:100,000 / 1:10,000; from Dr. Daniel Branton,
Harvard University, Cambridge, MA) or culture
Drosophila development without spectrin tetramerization
RESULTS
Characterizing the fly spectrin head-to-head
interactions - The α/β-spectrin H2H interaction
can be recapitulated using small two-repeat
fragments (Fig. 1A) of each subunit (35,36). We
cloned and expressed these fragments of the fly
α-, β- and βH-spectrin using homologous
endpoints (Table 1) (35). The α-specR22S construct
that was reported to have a Ts phenotype
contained a Myc tag at its N-terminus, but it was
not shown whether the presence of the tag was
important for this. This addition probably has a
minimal effect on the H2H interaction because it
resides at the end of an unstructured region Nterminal to the tetramerization site in α-spectrin
(35) and is fully functional in rescue experiments
(20,29,30). Nonetheless we felt it was important
to reproduce the previous construct exactly and
therefore included the tag. Solubility was good,
purity was high (Fig. 1B) and DSC experiments
(Fig. 1C) yielded melting profiles very comparable
to published spectrin repeat data (6,37) indicating
that they are in native conformation.
HPLC analysis indicates that α0-1 forms a
complex with both β16-17 and βH31-32 (Fig. 2A).
The Kd for these interactions was determined by
calorimetry (Fig. 2) (6) and indicates that the
wild-type interactions have a Kd (23oC) of 14nM
for α0-1/β16-17 and of 5 nM for α0-1/βH31-32
(Fig. 2C). These values are most similar to other
non-erythroid α/β interactions (5,6) rather than
that of the red cell where the Kd is 400-840 nM
(5,6). These results reinforce the notion that the
high Kd for red cell α1/βΙ-spectrin binding is a
special case.
In contrast, α0-1R22S binding to β16-17 is
not detectable (Fig. 2B) and binding to βH31-32 is
reduced ~1000 fold to 3.7-15µM (Fig. 2B,C),
across the reported permissive to non-permissive
5
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
temperature range for the α-specR22S phenotypes
(Fig. 2D) (24). While no H2H interaction between
α/β-spectrins seems likely in vivo, the residual
interaction between α0-1R22S and βH31-32 might
permit some tetramer formation depending upon
the local spectrin concentration. However, in the
red cell, which probably represents an extreme
high for an in vivo spectrin concentration, the
R28S substitution similarly reduces the Kd (6) and
has pathological consequences (38). So it seems
unlikely that the residual binding between
α0-1R22S and βH31-32 would sustain a wild-type
network in vivo. We conclude that the reported Ts
phenotype of the α-specR22S allele is not due to a
reversible H2H interaction.
α-spectrinR22S rescues α-spectrin null flies to
adulthood- Existing stocks expressing α-specR22S
were made in the mid 1990’s (24), no longer
express Myc-tagged α-spectrin and contain
promoter-deleted transgenes, suggesting that longterm constitutive expression of this protein has
been selected against in the stocks (not shown).
We therefore regenerated exactly the same
construct starting with the same full-length
α-spectrin cDNA (24) including the Myc tag,
which might participate in the reported
temperature sensitivity. To try to protect the
construct from eventual deletion, we immediately
crossed each new line into a background
heterozygous for the α-specrg41 null allele.
The crossing schemes shown in Figure 3 were
used to generate rescued flies. In this scheme flies
were produced that express up to 3 copies of the
transgene in an α-spectrin mutant background
(referred to as 1-3 copy rescue flies). Sibling flies
heterozygous for each mutant allele containing the
rescue construct are referred to as 1-3 copy
coexpression flies. Rescued flies were readily
obtained at 18, 25 and 29°C and with 1, 2 and in
some cases 3 copies of the transgene using the αα-specrg41/α-speclm51,
αspecrg41/α-speclm111,
rg41
lm32
rg41
R2
spec /α-spec
and α spec /Df(3l)R mutant
backgrounds. The third chromosome α-specR22S
transgene was sufficient to rescue to about 50% of
Mendelian expectation (55.4 ± 14.6%; n=7) while
the addition of the second chromosome transgene
raised this to almost 100% (98.3% ± 28.7%; n=3),
although the frequency was highly variable in both
cases. Flies were normal in morphology, and we
Samples were imaged on a CARV II spinning
disc confocal (BD Biosystems, Rockville, MD)
with a Retiga EXi camera (Q Imaging systems,
Surrey, BC) mounted on an Olympus BX50
microscope (Olympus America, Center Valley,
PA) and used iVision 4.5 software for control and
capture (Biovision, Exton PA). Figures were
assembled using Adobe CS4 Design Standard
(Adobe Systems Inc., San Jose, CA)
Drosophila development without spectrin tetramerization
our hands and are identical to chambers
degenerating in response to the nutritional
checkpoint (Fig. 5) (39). The existence of this
checkpoint and the importance of using well-fed
females to examine ovaries were not realized until
after Deng et al. published their paper (39). We
conclude that there is no such phenotype in the αspecR22S rescued females. However, there are more
subtle abnormalities in oogenesis not previously
described. First, we noticed that fusome structure
is altered. The fusome is a cytoplamic membrane
‘network’ that contains α/β-spectrin and Adducin
(40), forms at sites of incomplete cytokinesis,
associates with spindle poles during cystoblast
division, and reaches its greatest complexity in
germarium regions 2a/b before starting to
disappear. Staining for Adducin (1B1) reveals
fusome morphology (Fig. 6A) (see 41). In rescued
α-specrg41/α-speclm111 flies 1B1 staining becomes
fragmentary in region 2 (Fig. 6B-B’’) suggesting
that spectrin tetramerization is required for late
fusome integrity. We also see a similar
fragmentation when one copy of a wild-type αspectrin gene is present (Fig. 6C-C’) showing that
the α-specR22S can dominantly disrupt the fusome.
This is not a fixation artifact because
fragmentation was also seen with coexpression in
an α-spectrin GFP gene trap line when we imaged
freshly dissected germaria (Fig. 6D,D’). It is also
possible that expression of α-spectrin above wildtype levels is responsible for this phenotype. We
therefore overexpressed wild-type α-spectrin in
the germline in a wild-type α-spectrin background
using the Gal4/UAS system (42) with the MTDGal4
driver. Staining
MTD-Gal4>UAS-αspectrin37 ovarioles for 1B1 reveals fusomes of
normal complexity (Fig. 6E).
The second phenotype seen in the ovaries of
rescued females is an occasional gap in the follicle
cell monolayer (Fig. 6F-F’’). These are mostly
just one or two cell diameters in size, although a
handful of much larger ones have been observed.
Given the fertility of the rescued females and the
low frequency of degenerating chambers we
assume that most of these resolve themselves.
A number of other tissues are affected by loss
of function α-spectrin alleles in the fly. In
particular the spectrins are particularly necessary
in the nervous system (e.g. 27,43,44) and in the
6
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
do not see evidence of the sluggish behavior in
rescued flies reported by Deng et al. (24) (Movie
S1).
We speculate that the difference between our
results and the previous study by Deng et. al. (24)
may have arisen because of over- or underexpression from their original transgene
insertion(s), which would almost certainly have
had different levels of expression from our lines
due to position effects. We cannot do a direct
comparison since the original transgenic lines
were lost; however, we sought to verify that our
transgenes were producing α-specR22S at
physiological levels by comparison to wild-type
lines using quantitative immunoblots of whole fly
extracts (Fig. 4). The results appear to indicate that
our single and dual copy rescue flies express αspecR22S at ~80% wild-type levels and that
coexpression with one copy of wild-type αspectrin results in mild overexpression. However
these results are quite variable and do not achieve
statistical significance (One sample t-test versus
normalized control 0.07<P<0.21, n=7). Since two
copies of the transgene produce a better rescue
frequency while the size of the stable αspectrinR22S pool does not change, we suggest that
the rate of α-spectrin production may influence
viability. We conclude that our transgenes are
producing near wild-type amounts of α-specR22S
protein.
Probing with the anti-Myc antibody for the
tagged α-specR22S protein reveals that the presence
of wild-type α-spectrin often appears to cause the
selective destabilization of α-specR22S (Fig. 4C) or
that the ability to form tetramers results in the
selective stabilization of wild-type α-spectrin.
However, this result is also highly variable and
although striking on some blots, again does not
reach statistical significance (One sample t-test
versus normalized control 0.09<P<0.15, n=6).
α-spectrinR22S rescued α-spectrin null flies
exhibit subtle tissue abnormalities- Rescued males
and females raised at any temperature mated and
viable eggs are produced, indicating that early
development (prior to zygotic α-spectrin
transcription)
also
does
not
require
tetramerization. We examined the ovaries of
rescued flies closely because of the previously
reported degeneration phenotype in post-stage 7
egg chambers (24). Such chambers were rare in
Drosophila development without spectrin tetramerization
length proteins in vivo. Of course, it is formally
possible that in vivo conditions (e.g. local
concentration or interaction with other proteins)
could lead to tetramerization despite the presence
of the R22S substitution. However, the magnitude
of the effects of this mutation on tetramerization
are similar to that seen in the homologous
mutation in red cell spectrin, where the effective
spectrin concentration has been estimated to be as
high as 230mg/ml (~380µM) (47), and yet an
erythrocyte pathology still results. Therefore, the
simplest explanation for our results is that the
functionality of a conventional red cell-type
network is not that important in fly development in
any vital tissue.
Previous results suggesting that a conventional
network involving spectrin crosslinking of F-actin
might not be so important in non-erythroid tissues
came from examination of dominant H2Hdisrupting mutations in βΙ-spectrin that lead to
hereditary
elliptocytosis
and
hereditary
pyropoikillocytosis (see 48 and references therin).
Despite the expression of the non-erythroid βIΣII
spliceoform of this protein in other tissues, these
dominant effects are confined to the red cell.
Homozygosity for the βΙ-spectrinProvidence allele in
the tetramerization domain is lethal (49), but other
tissues, notably muscles, nonetheless develop
normally in affected individuals (50). However,
the issue of isoform redundancy could not be
addressed in this study since βIII- and βIVspectrins were unknown at the time.
In the fly, several papers suggest that βspectrin function in neurons and epithelial cells is
independent of α-spectrin and need not therefore
depend upon a conventional network (21,51,52).
Also in fly, the W2033R mutation in the βspectrin tetramerization domain was reported to
rescue β-spec mutant flies (53) although this was
not backed up with a biochemical demonstration
that the mutation had the homologous effect.
Vertebrate β-spectrin unaccompanied by αspectrin can be found in high molecular weight
complexes (54), but it is not clear whether these
are oligomers or complexes with other proteins. In
all these cases there is currently no published data
that indicates whether β-spectrin that is not bound
to α-spectrin is in any kind of network or is
operating in monomeric or oligomeric form.
DISCUSSION
The wild-type dissociation constants for the
tetramerization reaction between fly α/β-spectrin
and α/βH-spectrin are both in the low nM range
and about 100 fold lower than that of red cell
spectrin. This is in line with all other results for
non-erythroid spectrins to date (5), and is
consistent with the notion that the lower
tetramerization affinity associated with red cell
spectrin results from the specialized nature of this
cell type.
Our biochemical data strongly suggests that
tetramerization will not occur between αspectrinR22S and β-spectrin in vivo, and that the
interaction
is
severely
αR22S/βH-spectrin
compromised. The analysis of the recombinant
peptides used to determine the binding affinities
between the α-spectrin and β/βH spectrin
tetramerization domains represents an accepted
model since its inception over 20 years ago (45),
and analyses of larger proteolytic fragments or
full-length proteins has yielded the same
properties and binding affinities as the short
fragments used here (46). Furthermore, our results
are fully in line with other estimates of nonerythroid spectrin tetramerization interactions (5).
This strongly suggests that our measurements
reflect the tetramerization potential of the full-
7
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
midgut (34). We therefore performed initial
phenotypic analyses on the morphology of the
neuromuscular junction (NMJ) and brush border
of rescued third instar larvae. In NMJ of rescued
3rd instar larvae all our marker proteins (including
α-spectrin) localized normally (Fig. 6H-J). From
the location and complexity of the NMJ they
reveal, we further infer that the motor neurons find
and invade the musculature normally with little
difficulty. However, we do see that the NMJ itself
has a slightly altered morphology, with some of
the boutons in rescued larvae often being less
individuated (Fig. 6I,J). Similarly, phalloidin
staining of the brush border of the middle midgut
reveals no conspicuous abnormalities (Fig. 6 K,L).
The role of tetramerization at both of these
locations, is therefore not of sufficient importance
to compromise the tissue under culture conditions.
In future studies it will be interesting to see if any
specific functionalities at either of these locations
is perturbed by the absence of tetramerization.
Drosophila development without spectrin tetramerization
8
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
However, the selective advantage need only be
small over this period of time to result in the
present day high affinity interaction. It will be of
considerable interest for future studies to identify
what changes in functionality are present (e.g. at
the NMJ) in the rescued fly background that
underlie this selective pressure.
Our results change our perspective on these
essential proteins. The red cell model and its
variants in the terminal web and axons all depend
upon F-actin crosslinking by spectrin oligomers
that are mediated by the H2H interaction and are
widely assumed to apply in all non-erythroid
tissues. While there is a role for a network of this
type, our results clearly indicate that this is not
perhaps the core function of spectrin in nonerythroid tissue, and further reinforces the notion
that the dependence of the red cell on a fully
networked SBMS is a special case. Several studies
indicate a significant role for spectrin in
endomembrane trafficking (55-59). In this context
our results make sense, given that the size of most
transport organelles is far too small to
accommodate a traditional spectrin network.
It has been suggested that αΙ/βΙ-spectrin might
form oligomeric structures without the H2H
interaction (3) and it was recently shown that
human βH-spectrin can self associate (19). Such
results raise the possibility that non-conventional
networks may exist, and clearly their role would
not be tested by the current work.
The relative contribution of conventionally
networked spectrin to all the tissues in an
organism is directly assayed by this study for the
first time because all of the α-spectrin in the fly is
affected by the R22S substitution- there are no
other isoforms present. Since a viable and
functional adult is produced, H2H oligomerization
is by definition not a vital function, but since we
do detect minor morphological defects in several
tissues, it would appear that there is a role for the
conventional network, one of refinement. In the
fly’s normal environment, the role of H2H
oligomerization must be sufficiently significant to
provide a selective pressure for the maintenance of
this interaction. Since the structure of the
spectrins, including the tetramerization domains,
has been maintained for some 500 Myr (14), there
is clearly selective pressure for its maintenance.
Drosophila development without spectrin tetramerization
REFERENCES
1.
2.
3.
4.
5.
6.
7.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
9
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
8.
Elgasaeter, A., Stokke, B. T., Mikkelsen, A., and D., B. (1986) The molecular basis of
erythrocyte shape. Science 234, 1217-1222
Mohandas, N., and Gallagher, P. G. (2008) Red cell membrane: past, present, and future. Blood
112, 3939-3948
Morrow, J. S., and Marchesi, V. T. (1981) Self-assembly of spectrin oligomers in vitro: a basis
for a dynamic cytoskeleton. J. Cell Biol. 88, 463-468
Nans, A., Mohandas, N., and Stokes, D. L. (2011) Native ultrastructure of the red cell
cytoskeleton by cryo-electron tomography. Biophys. J. 101, 2341-2350
Bignone, P. A., and Baines, A. J. (2003) Spectrin alpha II and beta II isoforms interact with high
affinity at the tetramerization site. Biochem. J. 374, 613-624
Gaetani, M., Mootien, S., Harper, S., Gallagher, P. G., and Speicher, D. W. (2008) Structural and
functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin
tetramer site. Blood 111, 5712-5720
An X, L. M., Chasis JA, Mohandas N, Gratzer W. (2002) Shear-response of the spectrin dimertetramer equilibrium in the red blood cell membrane. J. Biol. Chem. 277, 31796-31800
Garbarz, M., Tse, W. T., Gallagher, P. G., Picat, C., Lecomte, M. C., Galibert, F., Dhermy, D.,
and Forget, B. G. (1991) Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a
kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping
due to a splice site mutation. J. Clin. Invest. 88, 76-81
Gallagher, P. G., Petruzzi, M. J., Weed, S. A., Zhang, Z., Marchesi, S. L., Mohandas, N.,
Morrow, J. S., and Forget, B. G. (1997) Mutation of a highly conserved residue of betaI spectrin
associated with fatal and near-fatal neonatal hemolytic anemia. J. Clin. Invest. 99, 267-277
Dubreuil, R. R., Byers, T. J., Stewart, C. T., and Kiehart, D. P. (1990) A beta-spectrin isoform
from Drosophila (beta H) is similar in size to vertebrate dystrophin. J. Cell Biol. 111, 1849-1858
Hirokawa, N., Cheney, R. E., and Willard, M. (1983) Location of a protein of the fodrin-spectrinTW260/240 family in the mouse intestinal brush border. Cell 32, 953-965
Wada, H., Kimura, K., Gomi, T., Sugawara, M., Katori, Y., Kakehata, S., Ikeda, K., and
Kobayashi, T. (2004) Imaging of the cortical cytoskeleton of guinea pig outer hair cells using
atomic force microscopy. Hear. Res. 187, 51-62
Xu, K., Zhong, G., and Zhuang, X. (2013) Actin, spectrin, and associated proteins form a periodic
cytoskeletal structure in axons. Science 339, 452-456
Thomas, G. H., Newbern, E. C., Korte, C. C., Bales, M. A., Muse, S. V., Clark, A. G., and
Kiehart, D. P. (1997) Intragenic duplication and divergence in the spectrin superfamily of
proteins. Mol. Biol. Evol. 14, 1285-1295
Morone, N., Fujiwara, T., Murase, K., Kasai, R. S., Ike, H., Yuasa, S., Usukura, J., and Kusumi,
A. (2006) Three-dimensional reconstruction of the membrane skeleton at the plasma membrane
interface by electron tomography. J. Cell Biol. 174, 851-862
Hartwig, J. H., and Yin, H. L. (1988) The organization and regulation of the macrophage actin
skeleton. Cell Motil. Cytoskeleton 10, 117-125
Morrow, J. S., Speicher, D. W., Knowles, W. J., Hsu, C. J., and Marchesi, V. T. (1980)
Identification of functional domains of human erythrocyte spectrin. Proc Natl Acad Sci U S A 77,
6592-6596
An, X., Guo, X., Yang, Y., Gratzer, W. B., Baines, A. J., and Mohandas, N. (2011) Inter-subunit
interactions in erythroid and non-erythroid spectrins. Biochim. Biophys. Acta 1814, 420-427
Papal, S., Cortese, M., Legendre, K., Sorusch, N., Dragavon, J., Sahly, I., Shorte, S., Wolfrum,
U., Petit, C., and El-Amraoui, A. (2013) The giant spectrin betaV couples the molecular motors to
phototransduction and Usher syndrome type I proteins along their trafficking route. Hum. Mol.
Genet. 22, 3773-3788
Drosophila development without spectrin tetramerization
20.
21.
22.
23.
24.
25.
26.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
10
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
27.
Lee, J. K., Coyne, R. S., Dubreuil, R. R., Goldstein, L. S., and Branton, D. (1993) Cell shape and
interaction defects in alpha-spectrin mutants of Drosophila melanogaster. J. Cell Biol. 123, 17971809.
Dubreuil, R. R., Wang, P., Dahl, S., Lee, J., and Goldstein, L. S. (2000) Drosophila beta spectrin
functions independently of alpha spectrin to polarize the Na,K ATPase in epithelial cells. J. Cell
Biol. 149, 647-656.
Thomas, G. H., Zarnescu, D. C., Juedes, A. E., Bales, M. A., Londergan, A., Korte, C. C., and
Kiehart, D. P. (1998) Drosophila betaHeavy-spectrin is essential for development and contributes
to specific cell fates in the eye. Development 125, 2125-2134
Austin, J., McKeown, C., and Patel, A. (1995) Sma-1 encodes a spectrin required for
morphogenesis of the C. elegans embryo. Mol. Biol. Cell 6, 270a
Deng, H., Lee, J. K., Goldstein, L. S., and Branton, D. (1995) Drosophila development requires
spectrin network formation. J. Cell Biol. 128, 71-79.
Harper, S. L., Begg, G. E., and Speicher, D. W. (2001) Role of terminal nonhomologous domains
in initiation of human red cell spectrin dimerization. Biochemistry 40, 9935-9943
Li, D., Harper, S., and Speicher, D. W. (2007) Initiation and propagation of spectrin heterodimer
assembly involves distinct energetic processes. Biochemistry 46, 10585-10594
Mazock, G. H., Das, A., Base, C., and Dubreuil, R. (2010) Transgene Rescue Identifies an
Essential Function for Drosophila {beta} Spectrin in the Nervous System and a Selective
Requirement for Ankyrin-2 Binding Activity. Mol. Biol. Cell
Sliter, T. J., Henrich, V. C., Tucker, R. L., and Gilbert, L. I. (1989) The genetics of the Dras3Roughened-ecdysoneless chromosomal region (62B3-4 to 62D3-4) in Drosophila melanogaster:
analysis of recessive lethal mutations. Genetics 123, 327-336
Lee, J. K., Brandin, E., Branton, D., and Goldstein, L. S. (1997) alpha-Spectrin is required for
ovarian follicle monolayer integrity in Drosophila melanogaster. Development 124, 353-362
de Cuevas, M., Lee, J. K., and Spradling, A. C. (1996) alpha-spectrin is required for germline cell
division and differentiation in the Drosophila ovary. Development 122, 3959-3968
Clyne, P. J., Brotman, J. S., Sweeney, S. T., and Davis, G. (2003) Green fluorescent protein
tagging Drosophila proteins at their native genomic loci with small P elements. Genetics 165,
1433-1441
Deng, H. (1995) Biochemical and Functional Characterization of Drosophila Spectrin Tetramer
Formation. Ph.D., Harvard University
Zarnescu, D. C., and Thomas, G. H. (1999) Apical spectrin is essential for epithelial
morphogenesis but not apicobasal polarity in Drosophila. J. Cell Biol. 146, 1075-1086.
Phillips, M. D., and Thomas, G. H. (2006) Brush border spectrin is required for early endosome
recycling in Drosophila. J. Cell Sci. 119, 1361-1370
Ipsaro, J. J., Harper, S. L., Messick, T. E., Marmorstein, R., Mondragon, A., and Speicher, D. W.
(2010) Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization
domain complex. Blood 115, 4843-4852
Kotula, L., DeSilva, T. M., Speicher, D. W., and Curtis, P. J. (1993) Functional characterization
of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site. J.
Biol. Chem. 268, 14788-14793
An, X., Zhang, X., Salomao, M., Guo, X., Yang, Y., Wu, Y., Gratzer, W., Baines, A. J., and
Mohandas, N. (2006) Thermal stabilities of brain spectrin and the constituent repeats of subunits.
Biochemistry 45, 13670-13676
Coetzer, T. L., Sahr, K., Prchal, J., Blacklock, H., Peterson, L., Koler, R., Doyle, J., Manaster, J.,
and Palek, J. (1991) Four different mutations in codon 28 of alpha spectrin are associated with
structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis. J. Clin.
Invest. 88, 743-749
Soller, M., Bownes, M., and Kubli, E. (1999) Control of oocyte maturation in sexually mature
Drosophila females. Dev. Biol. 208, 337-351
Drosophila development without spectrin tetramerization
40.
41.
42.
43.
44.
45.
46.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
11
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
47.
Lighthouse, D. V., Buszczak, M., and Spradling, A. C. (2008) New components of the Drosophila
fusome suggest it plays novel roles in signaling and transport. Dev. Biol. 317, 59-71
de Cuevas, M., and Spradling, A. C. (1998) Morphogenesis of the Drosophila fusome and its
implications for oocyte specification. Development 125, 2781-2789
Brand, A. H., and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates
and generating dominant phenotypes. Development 118, 401-415
Pielage, J., Fetter, R. D., and Davis, G. W. (2005) Presynaptic spectrin is essential for synapse
stabilization. Curr. Biol. 15, 918-928
Pielage, J., Fetter, R. D., and Davis, G. W. (2006) A postsynaptic spectrin scaffold defines active
zone size, spacing, and efficacy at the Drosophila neuromuscular junction. J. Cell Biol. 175, 491503
Kotula, L., DeSilva, T. M., Speicher, D. W., and Curtis, P. J. (1993) Functional characterization
of recombinant human red cell alpha- spectrin polypeptides containing the tetramer binding site.
J. Biol. Chem. 268, 14788-14793.
DeSilva, T. M., Peng, K. C., Speicher, K. D., and Speicher, D. W. (1992) Analysis of human red
cell spectrin tetramer (head-to-head) assembly using complementary univalent peptides.
Biochemistry 31, 10872-10878
Lux, S. E. (1979) Spectrin-actin membrane skeleton of normal and abnormal red blood cells.
Semin. Hematol. 16, 21-51
Gallagher, P. G., and Forget, B. G. (1996) Hematologically important mutations: spectrin variants
in hereditary elliptocytosis and hereditary pyropoikilocytosis. Blood Cells Mol. Dis. 22, 254-258
Gallagher, P. G., Weed, S. A., Tse, W. T., Benoit, L., Morrow, J. S., Marchesi, S. L., Mohandas,
N., and Forget, B. G. (1995) Recurrent fatal hydrops fetalis associated with a nucleotide
substitution in the erythrocyte beta-spectrin gene. J. Clin. Invest. 95, 1174-1182
Weed, S. A., Stabach, P. R., Oyer, C. E., Gallagher, P. G., and Morrow, J. S. (1996) The lethal
hemolytic mutation in beta I sigma 2 spectrin Providence yields a null phenotype in neonatal
skeletal muscle. Lab. Invest. 74, 1117-1129
Garbe, D. S., Das, A., Dubreuil, R. R., and Bashaw, G. J. (2007) beta-Spectrin functions
independently of Ankyrin to regulate the establishment and maintenance of axon connections in
the Drosophila embryonic CNS. Development 134, 273-284
Hulsmeier, J., Pielage, J., Rickert, C., Technau, G. M., Klambt, C., and Stork, T. (2007) Distinct
functions of alpha-Spectrin and beta-Spectrin during axonal pathfinding. Development 134, 713722
Das, A., Base, C., Dhulipala, S., and Dubreuil, R. R. (2006) Spectrin functions upstream of
ankyrin in a spectrin cytoskeleton assembly pathway. J. Cell Biol. 175, 325-335
Porter, G. A., Scher, M. G., Resneck, W. G., Porter, N. C., Fowler, V. M., and Bloch, R. J. (1997)
Two populations of beta-spectrin in rat skeletal muscle. Cell Motil. Cytoskeleton 37, 7-19
Tjota, M., Lee, S. K., Wu, J., Williams, J. A., Khanna, M. R., and Thomas, G. H. (2011) Annexin
B9 binds to beta(H)-spectrin and is required for multivesicular body function in Drosophila. J.
Cell Sci. 124, 2914-2926
Lorenzo, D. N., Li, M. G., Mische, S. E., Armbrust, K. R., Ranum, L. P., and Hays, T. S. (2010)
Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce
neurodegeneration in Drosophila. J. Cell Biol. 189, 143-158
Ayalon, G., Hostettler, J. D., Hoffman, J., Kizhatil, K., Davis, J. Q., and Bennett, V. (2011)
Ankyrin-B Interactions with Spectrin and Dynactin-4 Are Required for Dystrophin-based
Protection of Skeletal Muscle from Exercise Injury. J. Biol. Chem. 286, 7370-7378
Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007)
Ankyrin-G and beta2-spectrin collaborate in biogenesis of lateral membrane of human bronchial
epithelial cells. J. Biol. Chem. 282, 2029-2037
Drosophila development without spectrin tetramerization
59.
Kizhatil, K., Davis, J. Q., Davis, L., Hoffman, J., Hogan, B. L., and Bennett, V. (2007) AnkyrinG is a molecular partner of E-cadherin in epithelial cells and early embryos. J. Biol. Chem. 282,
26552-26561
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
12
Drosophila development without spectrin tetramerization
ACKNOWLEDGEMENTS
The authors would like to thank Peter Hembach for technical assistance, Julia Fecko for assistance
with calorimetry at Penn State, Dr. Ronen Marmorstein for emergency use of his calorimeter, Dr.
Lorraine Santy for use of her blot imager, members of the Thomas lab for critically reading the
manuscript, the Bloomington stock center and investigators who contributed antibodies and fly strains.
FIGURE LEGENDS
FIGURE 1. Characterization of expressed spectrin fragments. A. Schematic diagram illustrating the
location of the tetramerization site in an (αβ)2-spectrin tetramer (pink arrow) above the structure of the
vertebrate αI/βI-spectrin interaction at this location. The structure backbone is colored to illustrate the
spectrin repeats β16 (yellow), β17 (green), α0 (red) and α1 (blue). The location of the R28 side chain is
shown in space filling representation. B. SDS-PAGE analysis of purified proteins and markers. Although
the proteins do not migrate in the predicted order, MALDI analysis indicates that all the proteins have the
expected molecular weight (Table 1). C. DSC reveals typical spectrin repeat melting profiles (e.g. 6,37)
indicating correctly folding. Dashed red lines are fitted curves estimating the melting temperatures at:
59.6, 60.8, 49.0/61.4 and 48.6/75.2oC for α0-1wt, α0-1R22S, βH31-32, β16-17 respectively. DSC
experiments were done once, primarily for quality control purposes.
FIGURE 2. Complex formation between α- and β-spectrin tetramerization domains. A. HPLC gel
filtration profiles. Solid lines show an equimolar mixture of α0-1 and β16-17 (top) or βH31-32 (bottom);
dashed lines α0-1 alone; dotted lines β16-17 (top) and βH31-32 (bottom). Complex formation is robust in
both cases. B. Representative ITC analyses for binding between α0-1 (αwt) or α0-1α-specR22S (αR22S)
and β16-17 (β) or βH31-32 (βH) as indicated. C. Table showing the Kd (with 95% confidence intervals)
determined for each interaction at three different temperatures. n – number of replicates; UND –
interaction undetectable. The α-specR22S mutation eliminates interaction with β16-17 whereas a reduction
of ~1000 fold is seen with βH31-32.
FIGURE 3. Genetics of crosses resulting in a-specR22S rescued and coexpressing flies.
FIGURE 4. Analysis of α-spectrin levels by quantitative immunoblot. A. An example blot where fly
extracts from wild-type, α-spectrinR22S rescued (one or two copies of transgene) and α-spectrinR22S
coexpressing (one or two copies of transgene) flies were probed for α-spectrinR22S (via its myc-tag), total
α-spectrin and α-tubulin (for normalization). B. Comparison of total α-spectrin levels with wild-type.
13
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
FOOTNOTES
*This work was supported by grants MCB-1122013 to GHT from The National Science Foundation and
HL038794 to DWS from the National Institutes of Health
1
From The Department of Biology and The Department of Biochemistry and Molecular Biology
The Pennsylvania State University, University Park, PA, 16802
2
Systems Biology Division, The Wistar Institute, Philadelphia, PA, 19104
3
The Department of Biochemistry and Molecular Biology
Thomas Jefferson University, Philadelphia, PA, 19107 USA
4
To whom correspondence should be addressed: Graham H Thomas, Department of Biology, Department
of Biochemistry and Molecular Biology, 208 Mueller Laboratory, The Pennsylvania State University,
University Park, PA 16802, USA, Tel.: (814) 863-0716; email; Fax: (814) 865-9131; E-mail:
[email protected]
5
The abbreviations used are: SBMS – spectrin based membrane skeleton; H2H – head-to-head
tetramerization interaction; Ts – temperature sensitive; DSC – differential scanning calorimetry
Drosophila development without spectrin tetramerization
Rescued flies have ~80% of the α-spectrin seen in wild-type, while coexpressed α-spectrinR22S is not
additive with wild-type, probably reflecting the observation that it is destabilized in the presence of wildtype protein (panel C); C. Comparison of α-spectrinR22S levels in coexpressing and rescued flies. There is
less α-spectrinR22S when wild-type α-spectrin is present. Genotype rescued: a-specrg41/Df(3L)RR2.
Background for coexpression: a-specrg41/TM6B. Error bars represent 95% confidence intervals.
FIGURE 5. Degenerating egg chambers in α -specR22S coexpression and rescue females. Staining is
for Dlg or α-spectrin as cell shape markers and DAPI to show the characteristic pycnotic nuclei (e.g.
arrow in A). A, B. degenerating chambers (asterisks) from one copy coexpression females held at 22°C
and 29°C respectively for 4-5 days. C, D. degenerating chambers (asterisks) from one copy rescue
females held at 22°C and 29°C respectively for 4-5 days. (Note that the degenerating chamber in C
overlies a normal chamber). This phenotype is absolutely typical of degeneration triggered by the stage 8
nutritional checkpoint and occurs at a frequency of less than 10% in all genotypes examined as long as
females were well fed on fresh yeast paste for 4-5 days. Scale bars represent 20µm.
α
14
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
FIGURE 6. Tissue defects in α -specR22S rescued α-spec mutants. Ovaries: Germaria and egg chambers
stained with the 1B1 antibody (A-C’, E-G’) or expressing an α-spectrin GFP protein trap (D, D’).
Germarium images are maximum projections of confocal stacks, most of which were digitally tilted to
obtain the optimum view. A. Wild-type. Inset shows two fusomes showing the normal branching structure
in region 2b. One fusome is indicated by the dashed line. B-B’’. 1, 2 or 3 copy rescue α-speclm111/αspecrg41 flies (1cr, 2cr and 3cr respectively). C-C’. α-speclm111/+(TM6) heterozygous flies coexpressing 2
or 3 copies of α-specR22S (2cx and 3cx respectively). D-D’. αwt – weePα/TM6. α1cx – is weePα/P{αspec -specR22S} α-specRG41. E. Overexpression of wild-type α-spectrin (MTD-Gal4>UAS-α-spectrin37). FF’’. Egg chambers from α-specR22S rescued flies expressing 1, 2 or 3 copies of the α-specR22S transgene
stained with 1B1 that exhibit gaps in the normally continuous follicle cell monolayer (arrows, other labels
as in B). G-G’’. Germarium and egg chambers from two copy coexpression females stained for the Myc
epitope on the α-spectrinR22S protein, which can be seen to be present at the fusome and membrane in an
identical distribution to wild-type α-spectrin. Scale bars represent 10µm. Neuromuscular junctions
(NMJ): Anti-HRP (HRP) reveals the neuron. Synaptotagmin (SYT) marks the presynaptic side of the
boutons; α-spectrin (α-sp) is concentrated in the subsynaptic reticulum on the postsynaptic side of the
boutons. H-H’’- wild-type; I-J one copy rescue of α-specRG41/Df(3L)RR2. While rescued NMJ have many
regions that appear wild-type (arrows in I’) they often have regions where the boutons are less well
individuated (dashed lines in I’, J). As in the ovaries α-spectrinR22S exhibits a wild-type postsynaptic
accumulation at the boutons. Scale bars represent 20µm. Midgut: Staining for F-actin reveals the brush
borders on the apical domain of the cells. Wild-type cuprophilic cells have a deeply invaginated apical
surface with a thin channel out to the central lumen (arrowheads in K) but can also be more open
exhibiting a smooth domed surface (insets). Cuprophilc cells have more intense staining from longer
microvilli. L,M - in one copy rescue of α-specRG41/Df(3L)RR2 in these larvae there are large variations in
cell shape and position, with many malformed and crenellated cuprophilic cell lumens (asterisks);
however, a brush border is still present in all cases. Scale bars represent 20µm.
Drosophila development without spectrin tetramerization
TABLE 1. Spectrin fragments expressed and purified for this study.
Spectrin
fragment
Codons
From-To1
Primers (5’-3’)2
Mr
Predicted/
MALDI3
α 0-1
2…152
gspefENFT
…QQAL
gtgaattcGAGAACTTTACACCCAAAGAGGT
atgcggccgcttaGAGAGCCTGCTGCAGCTTTAGAC
18441.58/
18441.41
α myc01R22S
Myc…2…
R22S…152
gspefEQKL
…QQAL
gtgaattcGAGCAAAAGCTCATTTCTGAAGA
cgctcgagttaGAGAGCCTGCTGCAGCTTTAGAC
19702.0/
19700.14
β 16-17
1911…2093
gsTGDL
…RRQE
gtggatccACAGGCGACTTATTCCG
cgctcgagttaCTCCTGGCGCCTCTTCATTTCC
21651.61/
21637.03
gsAEQL
gtggatccGCAGAGCAGCTGCAGTCCTACTT
…QLEQ
cgctcgagttaCTGCTCCAGCTGGTGACGGAATA
βH 31-32
3412…3594
21666.50/
21666.80
15
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
Indicated are the relavant codons included by use of the indicated primers. Myc indicates that a Myc tag
was present on this construct. 1Amino acids in lower case were added by the construct and remain present
following thrombin cleavage. 2Bases in lower case were added for cloning purposes. The restriction sites
used (Eco RI, Not I and Xho I) are underlined in the primers and the added stop codon is in italics.
3
MALDI estimates of the molecular weights of the purified proteins
Drosophila development without spectrin tetramerization
Figure 1
50
40
30
20
15
10
kcal/mole
10
5
α0-1R22S
5
0
0
30 40 50 60 70 80 90 30 40 50 60 70 80 90
Temperature (oC)
Temperature (oC)
20
20
βH31-32
β16-17
15
15
kcal/mole
17
6β1 32
31
βH 22S
R
-1 wt
α0 0-1
α
B
α0-1wt
10
10
5
5
0
0
30 40 50 60 70 80 90 30 40 50 60 70 80 90
Temperature (oC)
Temperature (oC)
16
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
R28
10
kcal/mole
C
kcal/mole
A
Drosophila development without spectrin tetramerization
Figure 2
25
20
B
αwt β
αR22S β
α+β
mV
15
10
5
0
25
20
25
30
Elution time (min)
βH
35
α + βH
C
mV
15
10
5
0
25
30
Elution time (min)
35
17
αwt βH
αR22S βH
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
A
Drosophila development without spectrin tetramerization
Figure 3.
Parental genotypes
Relevant progeny genotypes
CyO, Cy
+
+
;
α−speclm111
TM6B, Tb Hu e
X
P[Ubi-α−specR22S]
CyO, Cy
;
α−specRG41 P[Ubi-α−specR22S]
TM6B, Tb Hu e





P[Ubi-α−specR22S]
+
CyO, Cy
+
;
α−specRG41 P[Ubi-α−specR22S]
TM6B, Tb Hu e
P[Ubi-α−specR22S]
;
TM6B, Tb Hu e
X
P[Ubi-α−specR22S]
CyO, Cy
;
α−speclm111
TM6B, Tb Hu e






α−speclm111
α−speclm111
TM6B, Tb Hu e
P[Ubi-α−specR22S]
CyO, Cy
CyO, Cy
α−specRG41 P[Ubi-α−specR22S]
;
+
α−specRG41 P[Ubi-α−specR22S]
α−speclm111
P[Ubi-α−specR22S]
+
P[Ubi-α−specR22S]
P[Ubi-α−specR22S]
;
α−specRG41 P[Ubi-α−specR22S]
P[Ubi-α−specR22S]
;
;
;
α−specRG41 P[Ubi-α−specR22S]
TM6B, Tb Hu e
α−specRG41 P[Ubi-α−specR22S]
α−speclm111
α−specRG41 P[Ubi-α−specR22S]
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
+
;
Name used in paper
(selection criteria)
One copy rescue
(1xP[w+], Cy Hu+)
Two copy rescue
(2xP[w+], Cy+ Hu+)
One copy coexpression
(1xP[w+], Cy Hu)
One copy coexpression
(1xP[w+], Cy Hu)
Two copy coexpession
(2xP[w+], Cy+ Hu)
Two copy rescue
(2xP[w+], Cy Hu+)
α−speclm111
Three copy rescue
(3xP[w+], Cy+ Hu+)
P[Ubi-α−specR22S]
CyO, Cy
;
α−speclm111
TM6B, Tb Hu e
One copy coexpression
(1xP[w+], Cy Hu)
P[Ubi-α−specR22S]
P[Ubi-α−specR22S]
;
α−speclm111
TM6B, Tb Hu e
Two copy coexpression
(2xP[w+], Cy+ Hu)
α−specRG41 P[Ubi-α−specR22S]
Two copy coexpression
(2xP[w+], Cy Hu)
P[Ubi-α−specR22S]
;
CyO, Cy
TM6B, Tb Hu e
P[Ubi-α−specR22S]
α−specRG41 P[Ubi-α−specR22S]
P[Ubi-α−specR22S]
18
;
TM6B, Tb Hu e
Three copy coexpession
(3xP[w+], Cy+ Hu)
Drosophila development without spectrin tetramerization
Figure 4
S
22 ue
αResc
r
S
22 xp
αR oe
c
S
22 ue
αR sc
re
S
22 xp
αRcoe
pe
-ty
ild
w
A
0
1
1
2
_
+
+
+
_
+
+
2
S g
22uin
αResc
r
protein detected
# transgenes
αR22S
Total α
protein
present
αR22S
wt α
+
B
Total α-spectrin
C
α-spectrinR22S
19
+
_
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
Tub
Drosophila development without spectrin tetramerization
Figure 5
1cx 22oC
A’
A’’
B’
B’’
*
Dlg
B
DNA
1cx 29oC
*
Dlg
DNA
C’
C
C’’
1cr 22oC
*
Dlg
DNA
D’
D
D’’
1cr 29oC
*
DNA
α-spec
20
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
A
Drosophila development without spectrin tetramerization
Figure 6
A
B
wt
1cr
F
H’
H
wt
K
HRP
B’’
C
2cr
3cr
2cx
F’
3cr
H’’
SYT
F’’
I
α-sp 1cr
L
3cr
wt
F-actin 1cr
*
D
D’
3cx
αwt
α1cx
G
M
*
21
2cx
α-sp SYT
*
F-actin 1cr
G’’
J
*
*
α OE
2cx
I’’
SYT
E
G’
2cx
I’
HRP
*
C’
*
F-actin
Downloaded from http://www.jbc.org/ by guest on November 12, 2014
2cr
B’