spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 10 January 2007
doi: 10.1242/dev.02758

Development 134, 713-722 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow All Versions of this Article:
dev.02758v1
134/4/713    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hülsmeier, J.
Right arrow Articles by Stork, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hülsmeier, J.
Right arrow Articles by Stork, T.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Distinct functions of {alpha}-Spectrin and ß-Spectrin during axonal pathfinding

Jörn Hülsmeier1, Jan Pielage1,*, Christof Rickert2, Gerd M. Technau2, Christian Klämbt1,{dagger} and Tobias Stork1

1 Institut für Neurobiologie, Badestr. 9, 48149 Münster, Germany.
2 Institut für Genetik, Saarstr. 21, 64123 Mainz, Germany.

{dagger} Author for correspondence (e-mail: klaembt{at}uni-muenster.de)

Accepted 27 November 2006


   SUMMARY
TOP
 SUMMARY
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell-shape changes during development require a precise coupling of the cytoskeleton with proteins situated in the plasma membrane. Important elements controlling the shape of cells are the Spectrin proteins that are expressed as a subcortical cytoskeletal meshwork linking specific membrane receptors with F-actin fibers. Here, we demonstrate that Drosophila karussell mutations affect ß-spectrin and lead to distinct axonal patterning defects in the embryonic CNS. karussell mutants display a slit-sensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals. Cell-type-specific rescue experiments demonstrate that ß-Spectrin is required autonomously and non-autonomously in cortical neurons to allow normal axonal patterning. Within the cell, ß-Spectrin is associated with {alpha}-Spectrin. We show that expression of the two genes is tightly regulated by post-translational mechanisms. Loss of ß-Spectrin significantly reduces levels of neuronal {alpha}-Spectrin expression, whereas gain of ß-Spectrin leads to an increase in {alpha}-Spectrin protein expression. Because the loss of {alpha}-spectrin does not result in an embryonic nervous system phenotype, ß-Spectrin appears to act at least partially independent of {alpha}-Spectrin to control axonal patterning.

Key words: Spectrin, Drosophila, Growth cone, Nervous system


   INTRODUCTION
TOP
 SUMMARY
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of multicellular animals is tightly linked to the evolution of a dynamic cell capable of forming and stabilizing manifold types of differentiation. This is most beautifully seen in the developing nervous system. Here, neurons migrate to their final destinations while, at the same time, the growth cones of the developing axons migrate towards their targets. In addition, dendritic specializations are formed and are kept in a dynamic equilibrium.

The dynamic changes of cell shape, as well as the subsequent stabilization of a specific form, demand a molecular machinery that can sense and transmit extracellular signals to the cytoskeleton. An important structural element that links cell adhesion proteins in the cell membrane to the F-actin cytoskeleton is the sub-membranous Spectrin network. Spectrins were first identified as important determinants defining the biconcave shape of erythrocytes. Erythrocytes that lack the Spectrin-based cytoskeleton loose their shape and stability, resulting in severe anemia in humans (Gallagher, 2004Go; Tse and Lux, 1999Go). Now, Spectrins are recognized as a large class of proteins ubiquitously expressed during development. The Spectrin proteins organize an extended protein network just below the plasma membrane by linking different actin fibers and many other proteins by numerous interaction motifs, such as the SH3 domain in {alpha}-Spectrin ({alpha}-Spec) (Bialkowska et al., 2005Go; Nedrelow et al., 2003Go). Furthermore, a pleckstrin-homology (PH) domain in ß-Spectrin (ß-Spec) allows its direct binding to membrane lipids (Williams et al., 2004Go). One of the best-characterized adaptor proteins that binds to Spectrin is Ankyrin, which can mediate interaction with other cellmembrane-associated receptors or channel proteins (Bennett and Chen, 2001Go; De Matteis and Morrow, 2000Go).

Generally, Spectrins are long rod-like structural proteins and have been found in all metazoan species. {alpha}- and ß-Spectrin form antiparallel dimers that associate in a head-to-head fashion to form an ({alpha}ß)2 hetero-tetramer (Bennett and Baines, 2001Go). In humans, Spectrins are found in all cells, and several different {alpha}- and ß-Spectrin isoforms exist (Bennett and Baines, 2001Go; Berghs et al., 2000Go; Dhermy, 1991Go). In Drosophila, only one {alpha}-spectrin gene, one ß-spectrin and one ßH-spectrin gene have been described (Dubreuil et al., 1989Go; Dubreuil et al., 1990Go; Lee et al., 1993Go; Thomas and Kiehart, 1994Go; Thomas et al., 1998Go). The Drosophila {alpha}- and ß-Spectrins share about 60% sequence identity with their human homologs, whereas the ßH-Spectrin isoform shares only 34% with its human homolog. ßH-Spectrin expression is found only in epithelial cells, whereas {alpha}- and ß-Spectrin are ubiquitously expressed during development.

Within Drosophila epithelia, the ({alpha}ß)2-Spectrin tetramer is found at basolateral membranes, whereas the ({alpha}ßH)2-Spectrin tetramer is localized to the apical membrane domain only (de Cuevas et al., 1996Go; Dubreuil et al., 1997Go; Lee et al., 1997Go; Pesacreta et al., 1989Go). At cell-cell contact zones, the basolateral ({alpha}ß)2-Spectrin is recruited to Neuroglian, a Drosophila homolog of the L1-cell adhesion molecule, via the adaptor protein Ankyrin. An additional protein recruited to the basolateral cell membrane is the Na+/K+-ATPase (Dubreuil et al., 1996Go; Dubreuil et al., 1997Go; Dubreuil et al., 2000Go; Nelson and Veshnock, 1987Go). A functional correlate of these interactions has recently been demonstrated in the neuromuscular junction. Here, reduction of both {alpha}- and ß-Spectrin not only leads to a mis-localization of Neuroglian and Fasciclin II, another cell adhesion protein, but finally results in a destabilization and retraction of the synapse (Featherstone et al., 2001Go; Pielage et al., 2005Go).

In Drosophila, mutations in all spectrin genes have been identified. Flies lacking ßH-spectrin function show reduced viability only and surviving flies exhibit relatively mild phenotypes, arguing against an essential function in determining epithelial-cell polarity (Zarnescu and Thomas, 1999Go). In contrast to ßH-spectrin, {alpha}-spectrin and ß-spectrin are both essential genes. Only 50% of the embryos lacking zygotic {alpha}-spectrin expression reach the larval stages, and the ones that do die in the first-instar stage (Dubreuil et al., 2000Go; Lee et al., 1993Go). The survival rate of homozygous-mutant ß-spectrin animals is even further reduced, and less than 10% of the mutant animals are able to leave the egg shells (Dubreuil et al., 2000Go). Clonal analyses have revealed essential functions of {alpha}-spectrin in the polarity of follicle cells (Lee et al., 1997Go; Thomas et al., 1998Go). Although {alpha}-Spectrin and ß-Spectrin are both found in a common protein complex, ß-spectrin-specific functions have been described (Dubreuil et al., 2000Go). Epithelial cells of the midgut lacking {alpha}-spectrin normally position the Na+/K+-ATPase in their cell membrane. By contrast, ß-spectrin mutants show an abnormal Na+/K+-ATPase distribution, suggesting that ß-Spectrin can function independent of {alpha}-Spectrin.

Both {alpha}- and ß-Spectrin are required during neuronal development. In C. elegans, it has been shown that ß-spectrin is required for normal axonal outgrowth and fasciculation (Hammarlund et al., 2000Go). In mammalian axons, the Spectrin proteins are required to stabilize transmembrane proteins at the nodes of Ranvier (Lacas-Gervais et al., 2004Go; Yang et al., 2004Go). In Drosophila, {alpha}- and ß-Spectrin have been shown to be involved in synapse organization and stability (Featherstone et al., 2001Go; Pielage et al., 2005Go). Here, we report the characterization of two mutants that were previously identified in a large phenotypic screen for genes affecting axonal pattern formation in the Drosophila embryo (Hummel et al., 1999aGo; Hummel et al., 1999bGo). We demonstrate that klötzchen and karussell (kus) encode {alpha}-Spectrin and ß-Spectrin proteins, respectively.

The kus (ß-spectrin) phenotype is characterized by a slit-sensitive crossing of the CNS midline by Fasciclin II-expressing axon fibers. Most prominently, we detected enlarged growth cones in single-cell analyses. Cell-type-specific genetic-rescue experiments demonstrated a requirement for ß-Spectrin in cortical neurons. This requirement, in part, may include non-autonomous effects, because single neurons cannot be rescued in homozygous-mutant kus embryos. We demonstrate that {alpha}-Spectrin protein levels are tightly coupled to the levels of ß-Spectrin by post-translational mechanisms, suggesting that {alpha}-spectrin mutants may share ß-spectrin-mutant phenotypes. However, within the nervous system, ß-Spectrin appears to act independently of {alpha}-Spectrin.

We further demonstrate that klötzchen mutants, which were initially selected based on a phenotype distinct from ß-spectrin mutants, affect the {alpha}-spectrin locus. We show that a reduction in {alpha}-Spectrin levels render the animal very sensitive to background mutations and temperature. klötzchen-mutant flies that do not carry a background mutation did not show a mutant CNS phenotype and, similarly, germline clones of hypomorphic alleles did not result in an abnormal nervous system phenotype. Thus, within the Drosophila nervous system, ß-spectrin appears to act independently of {alpha}-spectrin to stabilize neuronal growth cones.


   MATERIALS AND METHODS
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of antibodies
To generate ß-Spectrin-specific antibodies, we cloned 1686 bp starting from the ATG of ß-spectrin into pQE31. The resulting fusion protein was expressed and purified according to the manufacturer's instructions (Qiagen), and was used to immunize rabbits (Davids, Regensburg).

Generation of transgenes
To generate a UAS::ß-spectrin construct we used the cDNA clone AT24411 (BDGP), which contains the 5' region of the ß-spectrin mRNA. The 3' third of the mRNA was cloned via a RT-PCR approach (details are available on request). The resulting clones were subcloned in pUAST and the sequence was verified by sequencing. Subsequent germline transformation was performed according to standard procedures. Several independent insertion lines were tested, which all showed similar effects. An UAS::ß-spectrindsRNA construct was made using a 500 bp fragment from the 5' region of the ß-spectrin open reading frame (ORF). Several independent transgenic lines were established. Only lines that led to a lethal phenotype when crossed to daughterless-Gal4 were used in this study.

Genetic analyses
EMS mutagenesis of isogenic chromosomes has been described by Hummel et al. (Hummel et al., 1999aGo). All complementation analyses were performed at 25°C under standard conditions. The duplication Dp(1;3)BS3iD2 (provided by R. Dubreuil, University of Illinois, Chicago, IL), which rescues the lethality of five kus mutant chromosomes, was used. To remove the background lethal mutations from the {alpha}-spectrinE2-26 mutation, we first exchanged most of the third chromosome using the rucuca multi-marker chromosome. Subsequently, all recessive mutations of the rucuca marker were removed following recombination. The resulting E2-26 chromosome could be rescued to full viability using an ubi:{alpha}-spectrin mini-gene (Lee et al., 1993Go). The following alleles were used: {alpha}-speclm88, {alpha}-specrg41 (Lee et al., 1993Go); {alpha}-specE2-26, {alpha}-specD4-65, {alpha}-specN2-141, ß-specG113, ß-specE175, ß-specE292, ß-specH127, ß-specL105, ß-specM046, ß-specS012 (Hummel et al.1999aGo); {alpha}-specN-2, {alpha}-specP-2, {alpha}-specS-1, {alpha}-spec1.3, {alpha}-spec1.2.1 (this work); ß-specem6, ß-specem15, ß-specem21 (Dubreuil et al., 2000Go); slitB1-32 (Hummel et al., 1999aGo); and gcmP1 (Jones et al., 1995Go). To identify mutant animals, we employed GFP- or lacZ-labeled balancer chromosomes. The FRT elements ubi::GFP FRT19A and rsp174 P[white+]70C FRT80B were used, and mitotic recombination was induced by an ey::Flp transgene (Bloomington stock center). The following Gal4 strains were used: ptc::Gal4, da::Gal4, ap::Gal4, elav::Gal4 (Bloomington Stock Center); sim::Gal4 (Scholz et al., 1997Go); and repo::Gal4 (provided by B. Jones, University of Mississippi, USA).

Labeling and antibodies
Immunohistochemistry was performed as previously described (Hummel et al., 1999aGo). The following antibodies were used: mouse anti-Wrapper (Noordermeer, 1998Go); anti-{alpha}-Spectrin, anti-Repo, BP102, anti-Fasciclin II (Developmental Studies Hybridoma Bank); rabbit anti-ß-Galactosidase (Cappel); anti-myc 9E10 (Santa Cruz); anti-GFP (Invitrogen); anti-Kette (Bogdan and Klambt, 2003Go); anti-HRP-Cy5 (Dianova); and rabbit anti-ß-Spectrin (this work). DiI labeling of individual neuroblast cell clones was performed as previously described (Bossing and Technau, 1994; Bossing et al., 1996Go).


   RESULTS
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The karrussell-mutant phenotype
We have previously identified seven independently induced mutations in the Drosophila gene kus in a large-scale mutagenesis screen for defects in embryonic axon pattern formation (Hummel et al., 1999aGo; Hummel et al., 1999bGo). All kus mutants were characterized by their having a similar unique CNS phenotype that was not found in any other complementation group (Hummel et al., 1999aGo; Hummel et al., 1999bGo). In particular, stage-14 kus-mutant embryos were characterized by axon structures that loop out into the CNS cortex, a phenotype that was never observed in wild-type embryos (Fig. 1C). Interestingly, the circular axon pattern phenotype is resolved in older embryos and, at the end of embryogenesis, commissures appear to be fused, which is often indicative of CNS-midline defects (Klämbt et al., 1991Go). In addition, the longitudinal axon tracts are found in closer proximity to the CNS midline and are often thinner as compared to the wild type (Hummel et al., 1999aGo; Hummel et al., 1999bGo) (Fig. 1D). All kus mutants that have been identified lead to very similar mutant phenotypes.

karrussell mutations affect the ß-spectrin gene
We were able to rescue the lethality of five kus alleles using the chromosomal duplication Dp(1;3)BS3iD2, which affects the cytological interval 16A-D. The subsequent complementation assays that we performed showed that kus alleles cannot complement the lethality of the previously described ß-spectrin alleles em15 and em21 (Dubreuil et al., 2000Go). To further confirm that kus encodes ß-Spectrin, we sequenced the first 4000 bp of the ß-spectrin ORF in three mutant kus alleles and found a mutation resulting in a stop codon at position 538 of the deduced ß-spectrin ORF in the allele S012 (Fig. 2A). In addition, we assayed ß-Spectrin protein expression in homozygous-mutant kus embryos that were selected using a twist-GFP FM7 balancer chromosome. When using a newly generated antiserum against the N-terminus of ß-Spectrin, most kus mutants revealed altered ß-Spectrin protein expression in western blot experiments (Fig. 2B). The previously described allele em15 and our allele E292 encode proteins that presumably lack the C-terminal PH domain, but may retain the Ankyrin-binding domain. The strongest previously known ß-spectrin allele, em6, generates a 190 kDa large protein and thus may not be a complete null. The proteins encoded by G113, L015 and M046 are all significantly shorter compared with the wild-type protein. In the L015 mutant, the remaining protein appeared to encompass only two or three Spectrin repeats. Interestingly, all truncated ß-Spectrin proteins were relatively stable.


Figure 1
View larger version (153K):
[in this window]
[in a new window]

  		Fig. 1. The karussell phenotype. Dorsal view of dissected embryonic nervous systems stained for BP102 expression. Anterior is to the left. (A) In wild-type stage-14 embryos, commissures are established as regular axon fascicles. (B) In wild-type stage-16 embryos, the typical ladder-like axon pattern is established. Two clearly separated commissures (anterior commissure, ac; posterior commissure, pc) are found in every neuromere. Neuromeres are connected by the longitudinal connectives (lc). (C) In stage-14 hemizygous-mutant ß-spectrinkusS012 embryos, characteristic loops are found at the point where commissural axons normally enter the connectives (arrows). (D) Stage-16 hemizygous ß-spectrinkusS012 embryo. The longitudinal connectives are found closer to the midline and appear thinner (arrowhead); in addition, the segmental commissures are not clearly separated and appear slightly fused (arrow). (E,F) Hemizygous ß-spectrinkusS012 embryos that express ß-spectrin under the control of the elav promoter. The axonal phenotype is rescued.

 


Figure 2
View larger version (63K):
[in this window]
[in a new window]

  		Fig. 2. Molecular characterization of ß-spectrin mutants. (A) Schematic drawing of the 2291-amino acid large ß-Spectrin protein. The position of the S012 mutation is indicated. The bar indicates the part of the ß-Spectrin protein used for immunization. (B) Western blot of cell extracts derived from stage 15-17 embryos probed with a polyclonal antiserum generated against a ß-Spectrin fusion protein. First lane: wild type (wt), the 280 kDa large ß-Spectrin protein and a 200 kDa degradation product is visible. The 50 kDa band is a background band recognized by the polyclonal antibody. Mutant ß-spectrin embryos were selected using GFP-carrying balancer chromosomes. The genotype is indicated. In em6, em15, em21, G113, E292, L105 and M046 hemizygous embryos, truncated ß-Spectrin proteins are detected. E175, H127 and S012 hemizygous-mutant embryos lack detectable ß-Spectrin expression. (C) The same membrane was probed for expression of {alpha}-Spectrin. Notice the strong reduction of {alpha}-Spectrin expression in all genotypes, regardless of whether residual ß-Spectrin protein can still be detected or not. (D) Coomassie staining of same membrane to demonstrate equal loading. ABD, actin-binding domain; SR, spectrin repeats; Ank, ankykrin domain; PH, pleckstrin homology domain.

 
In protein extracts of hemizygous-mutant E175, H127 and S012 mutants, we could not detect any ß-Spectrin protein. Sequence analysis predicted that S012 mutants could generate a 20 kDa large protein fragment, which, however, was not detectable in western blot experiments. In summary, these data show that kus mutants affect the ß-spectrin locus. Despite these differences in ß-Spectrin protein sizes in the different kus and/or ß-spectrin mutations, we did not observe significant qualitative differences in the axonal phenotypes between these alleles. This indicates that a functional, full-length ß-Spectrin protein is required for normal nervous system development.

Neuronal expression of ß-spectrin rescues the karussell phenotype
It was previously shown that ß-Spectrin is expressed ubiquitously during Drosophila development (Dubreuil et al., 2000Go). Within the developing nervous system, ß-Spectrin is expressed in all neurons (see Fig. S1A in the supplementary material). Superficially, expression in the axonal compartment appeared somewhat lower when compared with the neuronal cell bodies. However, relatively high levels of ß- Spectrin expression can be found on specific axonal fascicles in both the connectives and the commissures (Fig. 3A, arrowheads). Within the commissures, the level of ß-Spectrin expression is further modulated and appears highest at the CNS midline (Fig. 3A, arrow). Co-expression with Wrapper, a specific marker for the midline glial cells (Noordermeer et al., 1998Go), demonstrated an overlap of this ß-Spectrin expression domain with the midline glial cells (see Fig. S1C,C' in the supplementary material). Unfortunately, the resolution of the confocal microscope did not allow us to determine whether the enhanced levels of ß-Spectrin expression at the midline were due to axonal or glial ß-spectrin expression.


Figure 3
View larger version (104K):
[in this window]
[in a new window]

  		Fig. 3. Expression of ß-Spectrin in the nervous system. Confocal views of dissected nervous systems of wild-type stage-16 embryos stained for expression of ß-Spectrin (white), axonal membranes (anti-HRP, green) or the SemaIIb{tau}Myc marker (red). (A) In wild-type embryos, ß-Spectrin expression is found in neuronal cell bodies and specific axonal fascicles (arrowheads). The arrow denotes increased levels of ß-Spectrin at the CNS midline. (B) In homozygous-mutant gcm embryos, lateral glial cells are absent. ß-Spectrin can still be detected at specific fascicles (arrowheads) and at the CNS midline (arrow). (C,C') The SemaIIb{tau}Myc marker is expressed by only one neuron per hemineuromere. This neuron is positioned at the lateral margin of the neuropil. SemaIIb{tau}Myc-positive axons project across the midline in the anterior commissure and then make a sharp turn to follow a specific path in the longitudinal connective. (D,D') In hemizygous-mutant kusS012 embryos, the SemaIIb{tau}Myc-positive neurons appear normally specified, but show irregular positions in the nerve cord, often being displaced towards the CNS midline. The SemaIIb{tau}Myc-positive fascicles are found closer to the CNS midline (double-headed arrow indicating distance in C and D) and the fascicle morphology appears changed. Often, ectopic projections are found that may correspond to enlarged growth cones (arrows). In addition, the precision in axonal pathfinding is lost; however, we did not observe ectopic crosses of the CNS midline.

 
Co-expression with the glial marker Repo (Campbell et al., 1994Go; Halter et al., 1995Go; Xiong et al., 1994Go) demonstrated that ß-Spectrin is also expressed in the longitudinal glia (see Fig. S1D,D' in the supplementary material). As glial processes invade the longitudinal connectives, ß-Spectrin expression within the longitudinal connectives might be due to glial expression. To discriminate between glial versus axonal expression, we analyzed gcm mutants, which have no lateral glial cells (Hosoya et al., 1995Go; Jones et al., 1995Go; Vincent et al., 1996Go). gcm-mutant embryos still showed a fasciculated ß-Spectrin staining pattern, indicating that axons and not glial cells are the major source for ß-Spectrin staining within the longitudinal connectives (Fig. 3A,B).

To further test whether expression in neurons or glia is required for the ß-spectrinkus axon phenotype, we performed cell-type-specific rescue experiments using Gal4/UAS-mediated expression of ß-spectrin. Ubiquitous expression of a UAS-ß-spectrin construct induced by a daughterless::Gal4 driver rescued hemizygous ß-spectrinkus-mutant animals to full viability (data not shown). Following expression of ß-spectrin in all lateral glial cells using a repo::Gal4 driver, we did not observe any alterations in the phenotypic strength (data not shown). Similarly, when ß-spectrin was expressed in all CNS-midline cells using the sim::Gal4 driver, the ß-spectrinkus-mutant phenotype was not rescued (data not shown). However, when we expressed ß-spectrin in all postmitotic neurons using the elav::Gal4 driver, we noted a complete rescue of the axonal patterning defects (Fig. 1). However, neuronal expression could not rescue the lethality associated with ß-spectrinkus mutants, demonstrating that ß-spectrin has additional essential functions outside of the nervous system (Dubreuil et al., 2000Go). To further test the requirement of ß-spectrinkus for Slit-Roundabout (Robo) signaling, we performed single-cell rescue experiments using both sim::Gal4 and apterous::Gal4 (ap::Gal4) driver strains. When ap::Gal4 was used to express ß-Spectrin no phenotypic rescue of their trajectory is observed in only a few cortical neurons and the ß-Spectrin-expressing fascicles are located in a unchanged position compared with the kus mutant (Fig. 4B-C'). Similarly, we failed to obtain cell-specific rescue in the MP1 fascicle following the expression of ß-spectrin using the sim::Gal4 driver (Fig. 4D-F').

These results suggest that ß-spectrinkus is required in cortical neurons for normal pathfinding and, furthermore, indicate that community effects are important in steering growth cones to their correct targets.

ß-Spectrin is required for axonal morphogenesis
To understand better how ß-Spectrin affects axonal pattern formation, we employed additional single-cell markers that reveal contralateral projections. In wild-type embryos, the SemaIIb:{tau}myc marker (Rajagopalan et al., 2000Go) is expressed in only one neuron per hemineuromere; this neuron projects its axon across the midline and the axon then follows a specific path within the longitudinal connectives (Fig. 3C,C'). In ß-spectrinkus mutants, specification of the SemaIIb neurons is not affected and the overall axonal trajectories are unchanged. However, the structure of the SemaIIb:{tau}myc-positive axon fascicle is severely altered in these mutants and the precision of axonal pathfinding is disrupted (Fig. 3D,D'). Although the normally straight axonal projection across the midline appeared irregular, ectopic crossings of the CNS midline were never observed (Fig. 3D,D'). In addition, the position of the SemaIIb:{tau}myc-expressing cell bodies was often shifted towards the CNS midline.

Very similar observations were made when analyzing the progeny of single labeled neuroblasts in stage-16 embryos (Fig. 5). Following the labeling of 210 individual DiI-labeled neuroblasts in 70 ß-spectrinkus- mutant embryos, we found mostly normal projection patterns and did not observe contralateral-projecting axons that ectopically crossed or illegitimately re-crossed the CNS midline. However, axons displayed abnormal varicosities and additional small, ectopic side-branches (Fig. 5). Most prominently, we noted alterations in the structure of the growth cones, which appeared enlarged with sometimes extensive, filopodia-like processes (Fig. 5A',C'; arrowheads). Thus, ß-Spectrin appears to be required to establish or maintain the structure of growth cones needed for precise axonal patterning.


Figure 4
View larger version (110K):
[in this window]
[in a new window]

  		Fig. 4. Non-autonomous effects of ß-spectrin on axonal patterning. Dissected preparations of stage-16 embryos stained for HRP (green) and ß-Galactosidase or ß-Spectrin, as indicated. (A,A') The ap::Gal4 driver directs {tau}lacZ expression into a small subset of ipsilateral-projecting neurons. (A') Notice the distance between the ß-Galactosidase-expressing fascicles (yellow bar). (B,B') Following expression of ß-spectrin in the ap::Gal4 pattern in kus mutants, the axonal trajectories are found at the same distance from the CNS midline as in hemizygous-mutant kus-mutant embryos (C,C'). (D,D') The sim::Gal4 driver directs {tau}lacZ expression into the MP1 neurons and some midline cells. Notice the distance of MP1 fascicles to the midline (yellow bar). (E,E') Following expression of ß-spectrin in the sim::Gal4 pattern in kus mutants, the MP1 axons are found at the same distance from the CNS midline as in hemizygous kus-mutant embryos (F,F').

 


Figure 5
View larger version (98K):
[in this window]
[in a new window]

  		Fig. 5. Morphology of the karussell-mutant neurons. Individual neuroblasts were filled using the DiI methodology. (A,E) DiI-labeled lineages of neuroblasts NB 3-5 and NB 7-3 in a wild-type embryo. (A') Higher magnification of the dotted area in A. The arrowhead denotes the growth cone. (B,F) Camera lucida drawings of wild-type lineages of NB 3-5 and NB 7-3. (C,G) DiI-labeled lineages of neuroblasts NB 3-5 and NB 7-3 in a hemizygous kus-mutant embryo. (C') Higher magnification of the dotted area in C. The arrowhead denotes the enlarged growth cone in the kus mutant. (D,H) Camera lucida drawings of these preparations reveal principally normal projection patterns, but altered fiber morphology and enlarged growth cones. No ectopic crossing of the CNS midline was observed.

 
ß-spectrinkus interacts with slit
A hallmark of the ß-spectrinkus phenotype is that the longitudinal connectives are located closer to the CNS midline in mature embryos (Hummel et al., 1999aGo; Hummel et al., 1999bGo) (Fig. 1). Correlating with the slight collapse at the CNS midline, we noted mild axon-crossing defects in hemizygous-mutant ß-spectrinkus animals. Whereas, in wild-type embryos, Fasciclin II-positive axon tracts never cross the CNS midline, we observed rare ectopic crosses of the midline in ß-spectrinkus-mutant embryos (Fig. 6). As midline crossing of Fasciclin II-positive axons is often sensitive to the dose of the midline repellent Slit, we performed gene-dosage experiments. Heterozygous slit-mutant animals, as well as ß-spectrinkus/+; sli/+ double-heterozygous female embryos, never showed ectopic crossing of Fasciclin II-positive axon tracts (Fig. 6). When we removed one copy of slit in a hemizygous ß-spectrinkus embryo, the number of neuromeres with ectopic midline crosses increased significantly (P<0.001; t-test), suggesting that kus function is somehow integrated in the Slitsignaling pathway (Fig. 5). Similar results were obtained for the kusG113 mutant, in which a truncated ß-Spectrin protein was expressed (Fig. 2; data not shown).


Figure 6
View larger version (90K):
[in this window]
[in a new window]

  		Fig. 6. ß-spectrinkus interacts with slit. (A) In wild-type stage-16 embryos, three distinct fascicles can be recognized following Fasciclin II staining. The most medial fascicle never crosses the CNS midline (see C for quantification). (B) In hemizygous-mutant ß-spectrinkus embryos, the inner fascicles appear to cross the midline (arrow). (D) Hemizygous-mutant kus embryo also lacking one copy of slit. The number of ectopic midline crosses increases significantly (arrows). (C) Quantification of the number of midline crosses of Fasciclin II-positive axon bundles. The number of embryos counted, the average number of ectopic crosses per embryo and the standard deviation for each genotype is indicated.

 


Figure 7
View larger version (126K):
[in this window]
[in a new window]

  		Fig. 7. ß-Spectrin regulates {alpha}-Spectrin stability. Top three panels show eye imaginal discs with homozygous-mutant cell clones lacking ß-Spectrin (red) or {alpha}-Spectrin (white), as indicated. Single confocal planes are shown. Clones were induced by expressing flp under the eyeless promoter and are marked by either the lack of GFP expression (green) or the loss of Spectrin expression. (A) In cell clones that are homozygous-mutant for the strong hypomorphic allele ß-spectrinG113, significantly reduced levels of ß-spectrin can be detected that appear to localize normally at the cell cortex. Concomitant to the reduction of ß-Spectrin expression we noticed a reduction in the level of {alpha}-Spectrin. (B) Similar results were obtained for cells homozygous for the ß-spectrinE292 mutation. (C) Clones homozygous for the strong {alpha}-spectrin allele D4-65 lack {alpha}-Spectrin protein expression but ß-Spectrin expression is not affected. Almost the entire eye is mutant for {alpha}-spectrin, except for a small area (arrow). (D) Wing imaginal disc expressing the UAS-ß-spectrin construct under the control of the patched::Gal4 driver. Elevated levels of ß-Spectrin result in a concomitant up-regulation in the level of {alpha}-Spectrin.

 
ß-spectrinkus mutations affect {alpha}-spectrin expression
In addition to affecting ß-spectrin expression, all of the ß-spectrinkus alleles affect {alpha}-spectrin protein levels (Fig. 2C). Interestingly, the effects on {alpha}-Spectrin levels do not correlate with the extent of the C-terminal ß-Spectrin deletions. This is in agreement with the notion that the C-terminal domain of ß-Spectrin is required to bind, and thus presumably stabilize, {alpha}-Spectrin (Deng et al., 1995Go; Yan et al., 1993Go). To determine the subcellular localization of {alpha}-Spectrin and the truncated ß-Spectrin proteins we stained whole-mount ß-spectrinkus-mutant embryos and, in addition, generated homozygous-mutant ß-spectrinkus tissue in the eye antennal imaginal discs using the Flp/FRT system. When we generated mutant clones using the two hypomorphic ß-spectrinkus alleles G113 or E292, we could detect reduced levels of ß-Spectrin expression that was still correctly localized at the cell membrane and a concomitant reduction in {alpha}-Spectrin protein levels (Fig. 7A,B). To test whether {alpha}-Spectrin is similarly required for the stability of ß-Spectrin protein, we generated homozygous-mutant eye discs lacking {alpha}-spectrin expression. In these experiments we observed the loss of {alpha}-Spectrin in the mutant clone but did not detect any change in the level of ß-Spectrin that was still correctly localized to the subcortical region of the cell (Fig. 7C). Thus, in contrast to {alpha}-Spectrin, ß-Spectrin can localize independently to the cell membrane. To further analyze the close regulatory interaction between these spectrin genes, we performed gain-of-function studies and expressed full-length ß-Spectrin in wild-type wing imaginal discs. This not only led to a clear up-regulation of ß-Spectrin protein expression but also to a concomitant increase in the levels of {alpha}-Spectrin protein (Fig. 7D). As RNA levels were not affected (data not shown), we conclude that there exists a post-translational mechanism stabilizing {alpha}-Spectrin protein. Together, these experiments demonstrate an intimate regulation of {alpha}- and ß-Spectrin, and show that ß-spectrinkus mutants are functional {alpha}- and ß-spectrin double mutants.


Figure 8
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 8. The {alpha}-spectrin gene is affected in klötzchen mutants. (A-F) CNS preparations of stage-16 embryos stained for BP102 (A-D) or ß-Spectrin (E,F). (A) Wild-type embryos are characterized by a regular axonal pattern with separated segmental commissures and longitudinal connectives. (B) Homozygous E2-26-mutant embryos show partially fused commissures. (C) A homozygous E2-26 mutant with no further background mutations shows no axonal phenotype. (D) When such embryos are allowed to develop at 4°C for 1 day, a fused-commissure phenotype develops. (E) ß-Spectrin protein in the ventral nerve cord of wild-type embryos. (F) In homozygous E2-26 mutants, ß-Spectrin expression in the neuropil appears altered. (G) {alpha}-Spectrin protein expression in some of the {alpha}-spectrin mutants. Proteins were isolated from ten stage 16/17 embryos and were separated on a 6% SDS gel. Following western blotting, {alpha}-Spectrin expression was detected using the monoclonal antibody 3A9. w1118 embryos were used as a wild-type control. If not otherwise indicated, homozygous mutants were used. Genotypes are as indicated. The P-2 mutation does not lead to a detectable truncation of {alpha}-Spectrin. In N-2 and, possibly, 1.3 mutants, a slight reduction of the {alpha}-Spectrin protein size is noted. In the deficiency Aprt32, and in the mutants rg41 and E2-26 all zygotic {alpha}-Spectrin expression is removed. Only maternal {alpha}-Spectrin expression is visible. (H) To control for equal loading, the same membrane was probed with anti-Kette antibodies.

 
{alpha}-spectrin is not required for axon pattern formation
Our data demonstrate that ß-spectrinkus-mutant embryos are impaired in both {alpha}-Spectrin and ß-Spectrin expression. In our recent large-scale EMS mutagenesis that led to the identification of ß-spectrinkus mutants, we identified several other mutations affecting the formation of the segmental commissures (Hummel et al., 1999aGo; Hummel et al., 1999bGo). One of the complementation groups identified was klötzchen, whose members show a commissural phenotype distinct to that of kus mutants (Hummel et al., 1999aGo) (Fig. 8B). Using standard genetic-mapping techniques, we localized the common lethality of five independent alleles to the chromosomal interval 61F-62A and subsequently showed that these mutants affect the {alpha}-spectrin locus. Sequence analysis of the klötzchen allele E2-26 revealed a A->T mutation at position 250 of the {alpha}-spectrin ORF that leads to the termination of translation after 83 amino acids (instead of 2416 amino acids for the full-length {alpha}-Spectrin protein). Thus, E2-26 probably represents a null allele. Interestingly, heterozygous {alpha}-spectrin-null mutations have relatively normal levels of {alpha}-Spectrin protein, indicating a strict regulation of the expression levels (Fig. 8G, compare lanes w1118 and Df(3L)aprt/+).

klötzchen mutants were initially isolated based on their embryonic CNS phenotype; however, following the removal of all lethal background mutations on the klötzchenE2-26 chromosome by meiotic recombination (see Materials and methods), no obvious axonal phenotypes were detected (Fig. 8C). Similarly, the previously described {alpha}-spectrin-null mutation rg41 (Lee et al., 1993Go) does not lead to an embryonic CNS phenotype. In addition, we did not see any Fasciclin II-positive axonal tracts crossing the CNS midline in homozygous E2-26 mutants (40 embryos scored, Fig. 6C). The distribution of ß-Spectrin protein in the longitudinal axon tracts did appear slightly altered, which might be due to fasciculation defects (Fig. 3A; Fig. 8E,F). Thus, zygotic {alpha}-Spectrin is not required for normal axonal patterning, but loss of {alpha}-spectrin renders neurons sensitive to background mutations. In agreement with this notion, we noted ectopic midline-crossing of Fasciclin II-positive axons when we removed one copy of slit in a homozygous {alpha}-spectrin-mutant background (Fig. 6C).

In an independent experiment that aimed to clarify the genetic organization at the genomic interval spanning the {alpha}-spectrin-discs lost region, we generated 21 additional EMS induced {alpha}-spectrin alleles. All mutants were lethal in trans to deficiencies of the region (Df(3L)Aprt32 or Df(3L)My10) and most animals died during the first-larval-instar stage. Two alleles, P-2 and 1.3, appeared to have normal levels of {alpha}-Spectrin protein (Fig. 8G) and were lethal during the early third-larval-instar stage, indicating that they are hypomorphic alleles. Interestingly, some of these {alpha}-spectrin mutants showed temperature-dependent intragenic complementation (see Table 1). Similarly, the axonal phenotype of {alpha}-spectrin alleles appeared temperature-sensitive (Fig. 8D). As the temperature sensitivity is not due to an altered stability of the {alpha}-Spectrin protein in these animals (see Fig. S2 in the supplementary material), the Spectrin network itself might be temperature labile.


View this table:
[in this window]
[in a new window]

 
 

   DISCUSSION
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The formation of the neuronal network requires a multitude of cellular interactions for precise axonal pathfinding and the establishment of specific synaptic connections (Dickson, 2002Go). In an attempt to identify some of the key regulators that control axonal pattern formation, we have isolated mutants that have specific defects in the developing embryonic nervous system of Drosophila (Hummel et al., 1999aGo; Hummel et al., 1999bGo). Here, we present the identification and further characterization of two of these mutations, kus and klötzchen.

Rescue experiments and direct sequence analysis demonstrate that kus encodes the Drosophila ß-Spectrin protein. kus mutants were initially isolated due to a distinct axonal phenotype, including ectopic CNS-midline crossing of Fasciclin II-positive axons. To further analyze this phenotype, we labeled the progeny of single neuroblasts in kus-mutant embryos but, despite the large number of labeled clones, we were unable to detect any aberrant midline crossings. Similarly, when we employed cell-type-specific Gal4 drivers, we could not see clear pathfinding defects across the midline. It is therefore likely that the observed phenotype is a result of inappropriate contact between medial Fasciclin II-expressing axons from both sides of the midline mimicking ectopic midline crossings. Because wild-type Slit levels are required to position the longitudinal fascicles, a reduction of slit gene dosage results, as expected, in a further medial positioning of the longitudinal connectives, explaining the increase in the number of ectopic midline crosses in these animals. Similar phenotypes were also observed by Garbe et al. (Garbe et al., 2007Go).

Interestingly, we found defects in the architecture of the neuronal growth cones in ß-spectrinkus-mutant animals, which may explain the general sensitivity of ß-spectrinkus-mutant neurons to guidance signals such as Slit. The enlarged growth cones detected in ß-spectrinkus mutants correlate nicely with data on growth cone formation after axotomy (Gitler and Spira, 1998Go); axonal injury leads to an increased activity of the protease calpain, which cleaves Spectrin and results in the removal of the submembranous Spectrin meshwork prior to the regeneration and growth of the growth cone (Gitler and Spira, 1998Go). In secretory cells, the submembranous Spectrin cytoskeleton prevents the premature fusion of vesicles with the plasma membrane (Aunis and Bader, 1988Go; Perrin et al., 1992Go). Similarly, Spectrins may function to regulate the fusion of intracellular membrane vesicles needed to enlarge and advance the growth cone (Gitler and Spira, 1998Go), which could explain the enlarged growth cones that we detected in ß-spectrin mutants (Fig. 4).

Within the Drosophila nervous system, {alpha}- and ß-Spectrin are the only Spectrins that are expressed. These two proteins form a heterodimer in which ß-Spectrin appears to be the key determinant, because {alpha}-Spectrin protein is only stable in the presence of ß-Spectrin and ectopic expression of ß-Spectrin leads to a concomitant increase in the level of {alpha}-Spectrin protein. To test whether this regulation occurs at the level of RNA or protein, we determined the expression of the corresponding transcripts, but noted no alteration (data not shown). It is possible that the association of {alpha}- and ß-Spectrin blocks ubiquitination of {alpha}-Spectrin and its subsequent degradation via the proteasome. Ubiquitination has been previously reported for {alpha}-Spectrin (Corsi et al., 1995Go; Galluzzi et al., 2001Go) and may thus help to define the correct protein-expression levels.

Despite the intimate coupling of the two expression profiles, it has been demonstrated that ß-spectrin can function independently of {alpha}-Spectrin. During the development of the midgut, the correct localization of the Na+/K+-ATPase requires only ß-spectrin, but not {alpha}-spectrin, function (Dubreuil et al., 2000Go). Similarly, the phenotypes associated with the different spectrin mutants isolated in this study are distinct. Whereas ß-spectrin leads to a typical looping of CNS axons during stage 13, no abnormal axonal phenotypes could be detected for {alpha}-spectrin alleles. Similarly, we failed to detect any midline phenotypes for Fasciclin II-positive axons in {alpha}-spectrinE2-26-null mutants. A possible explanation to this phenotypic discrepancy may be the maternal contribution of {alpha}-spectrin; however, a similarly strong maternal component has been described for ß-spectrin. Attempts to generate {alpha}-spectrin germline clones using the null allele rg41 failed because of an essential function of {alpha}-spectrin during oogenesis (de Cuevas et al., 1996Go). To circumvent this maternal {alpha}-spectrin function, we employed the hypomorphic {alpha}-spectrin-mutant alleles {alpha}-specN-2, {alpha}-specP-2 or a-spec1.3 to generate germline clones using the ovoD/FRT system. However, embryos with both impaired maternal and zygotic {alpha}-spectrin expression displayed no nervous system phenotype, supporting the notion that ß-spectrin acts independent of {alpha}-Spectrin protein.

{alpha}-spectrin mutations turned out to be sensitive to background mutations and temperature effects. In addition to the phenotypic effects of uncharacterized background mutations, we detected a temperature dependence of the spectrin-mutant phenotypes and temperature-dependent intragenic complementation of hypomorphic {alpha}-spectrin alleles. It is well-known that microtubule dynamics depend on temperature and that microtubules depolymerize in the cold (Osborn and Weber, 1976Go). As microtubule stability is already compromised in {alpha}-spectrin mutants (Pielage et al., 2005Go), any further destabilization might have significant effects on (neuronal) development (Dent and Gertler, 2003Go). Alternatively, temperature sensitivity might reflect differences in the efficacy of endocytosis. Although we cannot pinpoint the molecular mechanism underlying the temperature sensitivity of spectrin mutants, we can conclude that Spectrins act as a global stabilizing protein network that coordinates a large variety of membrane receptors, including the Robo receptor that is needed to sense the Slit protein.

Supplementary material
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/134/4/713/DC1


   ACKNOWLEDGMENTS
 
We thank G. Bashaw for communication and discussion of results prior to publication; and L. Goldstein, B. Dickson, R. Dubreuil, D. Branton, B. Jones and the Bloomington Stock Center for sending flies and antibodies. J.P. was supported by a pre-doctoral grant of the Boehringer-Ingelheim foundation. This work was funded by grants of the DFG (SPP 1111).


   Footnotes
 
* Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA. Back


   REFERENCES
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


Aunis, D. and Bader, M. F. (1988). The cytoskeleton as a barrier to exocytosis in secretory cells. J. Exp. Biol. 139,253 -266.[Abstract/Free Full Text]
Bennett, V. and Baines, A. J. (2001). Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 81,1353 -1392.[Abstract/Free Full Text]
Bennett, V. and Chen, L. (2001). Ankyrins and cellular targeting of diverse membrane proteins to physiological sites. Curr. Opin. Cell Biol. 13, 61-67.[CrossRef][Medline]
Berghs, S., Aggujaro, D., Dirkx, R., Jr, Maksimova, E., Stabach, P., Hermel, J. M., Zhang, J. P., Philbrick, W., Slepnev, V., Ort, T. et al. (2000). betaIV spectrin, a new spectrin localized at axon initial segments and nodes of ranvier in the central and peripheral nervous system. J. Cell Biol. 151,985 -1002.[Abstract/Free Full Text]
Bialkowska, K., Saido, T. C. and Fox, J. E. (2005). SH3 domain of spectrin participates in the activation of Rac in specialized calpain-induced integrin signaling complexes. J. Cell Sci. 118,381 -395.[Abstract/Free Full Text]
Bogdan, S. and Klambt, C. (2003). Kette regulates actin dynamics and genetically interacts with Wave and Wasp. Development 130,4427 -4437.[Abstract/Free Full Text]
Bossing, T., Udolph, G., Doe, C. Q. and Technau, G. M. (1996). The embryonic central nervous system lineages of Drosophila melanogaster. I. Neuroblast lineages derived from the ventral half of the neuroectoderm. Dev. Biol. 179, 41-64.[CrossRef][Medline]
Campbell, G., Goring, H., Lin, T., Spana, E., Andersson, S., Doe, C. Q. and Tomlinson, A. (1994). RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development 120,2957 -2966.[Abstract]
Corsi, D., Galluzzi, L., Crinelli, R. and Magnani, M. (1995). Ubiquitin is conjugated to the cytoskeletal protein alpha-spectrin in mature erythrocytes. J. Biol. Chem. 270,8928 -8935.[Abstract/Free Full Text]
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.[Abstract]
De Matteis, M. A. and Morrow, J. S. (2000). Spectrin tethers and mesh in the biosynthetic pathway. J. Cell Sci. 113,2331 -2343.[Abstract]
Deng, H., Lee, J. K., Goldstein, L. S. and Branton, D. (1995). Drosophila development requires spectrin network formation. J. Cell Biol. 128, 71-79.[Abstract/Free Full Text]
Dent, E. W. and Gertler, F. B. (2003). Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40,209 -227.[CrossRef][Medline]
Dhermy, D. (1991). The spectrin super-family. Biol. Cell 71,249 -254.[Medline]
Dickson, B. J. (2002). Molecular mechanisms of axon guidance. Science 298,1959 -1964.[Abstract/Free Full Text]
Dubreuil, R. R., Byers, T. J., Sillman, A. L., Bar-Zvi, D., Goldstein, L. S. and Branton, D. (1989). The complete sequence of Drosophila alpha-spectrin: conservation of structural domains between alpha-spectrins and alpha-actinin. J. Cell Biol. 109,2197 -2205.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]
Dubreuil, R. R., MacVicar, G., Dissanayake, S., Liu, C., Homer, D. and Hortsch, M. (1996). Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites. J. Cell Biol. 133,647 -655.[Abstract/Free Full Text]
Dubreuil, R. R., Maddux, P. B., Grushko, T. A. and MacVicar, G. R. (1997). Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly. Mol. Biol. Cell 8,1933 -1942.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]
Featherstone, D. E., Davis, W. S., Dubreuil, R. R. and Broadie, K. (2001). Drosophila alpha- and beta-spectrin mutations disrupt presynaptic neurotransmitter release. J. Neurosci. 21,4215 -4224.[Abstract/Free Full Text]
Gallagher, P. G. (2004). Hereditary elliptocytosis: spectrin and protein 4.1R. Semin. Hematol. 41,142 -164.[CrossRef][Medline]
Galluzzi, L., Paiardini, M., Lecomte, M. C. and Magnani, M. (2001). Identification of the main ubiquitination site in human erythroid alpha-spectrin. FEBS Lett. 489,254 -258.[CrossRef][Medline]
Garbe, D. S., Das, A., Dubreuil, R. R. and Bashaw, G. J. (2007). ß-Spectrin functions independently of Ankyrin to regulate the establishment and maintenance of axon connections in the Drosophila embryonic CNS. Development 134,273 -284.[Abstract/Free Full Text]
Gitler, D. and Spira, M. E. (1998). Real time imaging of calcium-induced localized proteolytic activity after axotomy and its relation to growth cone formation. Neuron 20,1123 -1135.[CrossRef][Medline]
Halter, D. A., Urban, J., Rickert, C., Ner, S. S., Ito, K., Travers, A. A. and Technau, G. M. (1995). The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster.Development 121,317 -332.[Abstract]
Hammarlund, M., Davis, W. S. and Jorgensen, E. M. (2000). Mutations in beta-spectrin disrupt axon outgrowth and sarcomere structure. J. Cell Biol. 149,931 -942.[Abstract/Free Full Text]
Hosoya, T., Takizawa, K., Nitta, K. and Hotta, Y. (1995). glial cells missing: a binary switch between neuronal and glial determination in Drosophila. Cell 82,1025 -1036.[CrossRef][Medline]
Hummel, T., Schimmelpfeng, K. and Klambt, C. (1999a). Commissure formation in the embryonic CNS of Drosophila. Development 126,771 -779.[Abstract]
Hummel, T., Schimmelpfeng, K. and Klambt, C. (1999b). Commissure formation in the embryonic CNS of Drosophila. Dev. Biol. 209,381 -398.[CrossRef][Medline]
Jones, B. W., Fetter, R. D., Tear, G. and Goodman, C. S. (1995). glial cells missing: a genetic switch that controls glial versus neuronal fate. Cell 82,1013 -1023.[CrossRef][Medline]
Klämbt, C., Jacobs, J. R. and Goodman, C. S. (1991). The midline of the Drosophila central nervous system: a model for the genetic analysis of cell fate, cell migration, and growth cone guidance. Cell 64,801 -815.[CrossRef][Medline]
Lacas-Gervais, S., Guo, J., Strenzke, N., Scarfone, E., Kolpe, M., Jahkel, M., De Camilli, P., Moser, T., Rasband, M. N. and Solimena, M. (2004). BetaIVSigma1 spectrin stabilizes the nodes of Ranvier and axon initial segments. J. Cell Biol. 166,983 -990.[Abstract/Free Full Text]
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. 123,1797 -1809.[Abstract/Free Full Text]
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.[Abstract]
Nedrelow, J. H., Cianci, C. D. and Morrow, J. S. (2003). c-Src binds alpha II spectrin's Src homology 3 (SH3) domain and blocks calpain susceptibility by phosphorylating Tyr1176. J. Biol. Chem. 278,7735 -7741.[Abstract/Free Full Text]
Nelson, W. J. and Veshnock, P. J. (1987). Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells. Nature 328,533 -536.[CrossRef][Medline]
Noordermeer, J. N., Kopczynski, C. C., Fetter, R. D., Bland, K. S., Chen, W. Y. and Goodman, C. S. (1998). Wrapper, a novel member of the Ig superfamily, is expressed by midline glia and is required for them to ensheath commissural axons in Drosophila.Neuron 21,991 -1001.[CrossRef][Medline]
Osborn, M. and Weber, K. (1976). Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane. Proc. Natl. Acad. Sci. USA 73,867 -871.[Abstract/Free Full Text]
Perrin, D., Moller, K., Hanke, K. and Soling, H. D. (1992). cAMP and Ca(2+)-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton. J. Cell Biol. 116,127 -134.[Abstract/Free Full Text]
Pesacreta, T. C., Byers, T. J., Dubreuil, R., Kiehart, D. P. and Branton, D. (1989). Drosophila spectrin: the membrane skeleton during embryogenesis. J. Cell Biol. 108,1697 -1709.[Abstract/Free Full Text]
Pielage, J., Fetter, R. D. and Davis, G. W. (2005). Presynaptic spectrin is essential for synapse stabilization. Curr. Biol. 15,918 -928.[CrossRef][Medline]
Rajagopalan, S., Vivancos, V., Nicolas, E. and Dickson, B. J. (2000). Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103,1033 -1045.[CrossRef][Medline]
Scholz, H., Sadlowski, E., Klaes, A. and Klambt, C. (1997). Control of midline glia development in the embryonic Drosophila CNS. Mech. Dev. 64,137 -151.[CrossRef][Medline]
Thomas, G. H. and Kiehart, D. P. (1994). Beta heavy-spectrin has a restricted tissue and subcellular distribution during Drosophila embryogenesis. Development 120,2039 -2050.[Abstract]
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.[Abstract]
Tse, W. T. and Lux, S. E. (1999). Red blood cell membrane disorders. Br. J. Haematol. 104, 2-13.[CrossRef][Medline]
Vincent, S., Vonesch, J. L. and Giangrande, A. (1996). Glide directs glial fate commitment and cell fate switch between neurones and glia. Development 122,131 -139.[Abstract]
Williams, J. A., MacIver, B., Klipfell, E. A. and Thomas, G. H. (2004). The C-terminal domain of Drosophila (beta) heavy-spectrin exhibits autonomous membrane association and modulates membrane area. J. Cell Sci. 117,771 -782.[Abstract/Free Full Text]
Xiong, W. C., Okano, H., Patel, N. H., Blendy, J. A. and Montell, C. (1994). repo encodes a glial-specific homeo domain protein required in the Drosophila nervous system. Genes Dev. 8,981 -994.[Abstract/Free Full Text]
Yan, Y., Winograd, E., Viel, A., Cronin, T., Harrison, S. C. and Branton, D. (1993). Crystal structure of the repetitive segments of spectrin. Science 262,2027 -2030.[Abstract/Free Full Text]
Yang, Y., Lacas-Gervais, S., Morest, D. K., Solimena, M. and Rasband, M. N. (2004). BetaIV spectrins are essential for membrane stability and the molecular organization of nodes of Ranvier. J. Neurosci. 24,7230 -7240.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:


Home page
 DevelopmentHome page
T. Stork, S. Thomas, F. Rodrigues, M. Silies, E. Naffin, S. Wenderdel, and C. Klambt
Drosophila Neurexin IV stabilizes neuron-glia interactions at the CNS midline by binding to Wrapper
Development, April 15, 2009; 136(8): 1251 - 1261.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Das, C. Base, D. Manna, W. Cho, and R. R. Dubreuil
Unexpected Complexity in the Mechanisms That Target Assembly of the Spectrin Cytoskeleton
J. Biol. Chem., May 2, 2008; 283(18): 12643 - 12653.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. Silies, Y. Yuva, D. Engelen, A. Aho, T. Stork, and C. Klambt
Glial Cell Migration in the Eye Disc
J. Neurosci., November 28, 2007; 27(48): 13130 - 13139.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Summary Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow All Versions of this Article:
dev.02758v1
134/4/713    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hülsmeier, J.
Right arrow Articles by Stork, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hülsmeier, J.
Right arrow Articles by Stork, T.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?