Abstract
Summary statement Ectotherms thrive within an often remarkable temperature range. At low temperature, betaTub97EF, a beta-tubulin paralog stabilizing microtubules, is upregulated in a tissue-specific manner in the fly Drosophila melanogaster.
Abstract Cells in ectotherms function normally within an often wide temperature range. As temperature dependence is not uniform across all the distinct biological processes, acclimation presumably requires complex regulation. The molecular mechanisms coping with the disruptive effects of temperature variation are still poorly understood. Interestingly, one of five different beta-tubulin paralogs, betaTub97EF, was among the genes up-regulated at low temperature in cultured Drosophila cells. As microtubules are known to be cold-sensitive, we analyzed whether betaTub97EF protects microtubules at low temperatures. During development at the optimal temperature (25°C), betaTub97EF was expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, expression was increased at low temperature (14°C). While betaTub97EF mutants were viable and fertile at 25°C, their sensitivity within the well-tolerated range was slightly enhanced during embryogenesis specifically at low temperatures. Changing beta-tubulin isoform ratios in hemocytes demonstrated that beta-Tubulin 97EF has a pronounced microtubule stabilizing effect. Moreover, betaTub97EF is required for normal microtubule stability in the gut. These results suggest that betaTub97EF up-regulation at low temperature contributes to acclimation by stabilizing microtubules.
Introduction
Many ecological niches are exposed to ambient temperature fluctuations. Distinct strategies have evolved to cope with the disruptive effects of temperature changes on cellular homeostasis. Endotherms like humans rely primarily on internally generated heat for maintenance of a relatively high and constant core body temperature. However, the majority of organisms are ectotherms. Their internal temperature is primarily dictated by the environment. But even in endotherms, the exposed peripheral cells need to function over a range of temperatures.
Arguably the most extensive analyses of the cellular response to low temperature in eukaryotes so far have been done with yeast (Gasch et al., 2000; Taymaz-Nikerel et al., 2016), confirming the general notion that rapid temperature shifts to nonlethal low temperature disrupt cellular homeostasis, triggering an initial transient stress response followed by persistent acclimation. Moreover, the response to low temperature is quite distinct from the general stress response induced by many other perturbations.
In animals, low temperature acclimation includes additional response levels (Denlinger and Lee, 2010; Hayward et al., 2007). Organ systems govern complex behavioral and metabolic responses. At the cellular level, microtubules have long been recognized to be cold-sensitive in mammals (Correia and Williams, 1983). Microtubules are dynamic cytoskeletal elements involved in innumerable processes (Nogales and Zhang, 2016). They are polymerized from heterodimers of alpha- and beta-tubulin. Heterodimers can associate longitudinally into protofilaments. Lateral interactions between parallel protofilaments organize these into hollow cylindrical tubes. Both alpha- and beta-tubulin bind GTP at a conserved site. GTP-tubulin heterodimers incorporate efficiently at growing microtubule ends. Polymerization is coupled to hydrolysis of the GTP bound to beta-tubulin. This regulates microtubule dynamics and gives rise to dynamic instability where growing phases alternate with shrinking phases (Mitchison and Kirschner, 1984; Zhang et al., 2015). Above a critical concentration, GTP-tubulin purified from mammalian sources polymerizes into microtubules at 37°C, largely driven by the entropically favorable displacement of structured water at the subunit interfaces (Correia and Williams, 1983). Reflecting the positive correlation between entropic drive and temperature, these microtubules depolymerize after a shift to 4°C in a GTP hydrolysis-independent manner (Fygenson et al., 1994).
Psychrophilic ectotherms assemble microtubules even below 0°C. Analysis of their genes have revealed amino acid changes that likely alter the temperature dependence of microtubule stability (Chiappori et al., 2012; Detrich et al., 2000; Modig et al., 2000; Tartaglia and Shain, 2008). While such evolutionary adaptations support life at constant low temperature, organisms exposed to short term thermal fluctuations presumably depend on temperature controlled regulation of microtubule dynamics. In principle, regulated expression of tubulin paralogs with distinct properties is conceivable, as well as control by posttranslational modifications and indirectly via microtubule-associated proteins (MAPs) and motors. Paralogs encoding distinct alpha- and beta-tubulins have evolved in many lineages (Findeisen et al., 2014). Sequence differences are found predominantly in the C-terminal tails, where also most posttranslational modifications occur. These modifications are thought to generate a ‘tubulin-code’ controlling binding and function of many MAPs and motors (Gadadhar et al., 2017; Janke, 2014; Sirajuddin et al., 2014; Yu et al., 2015).
In mammals, MAP6 adapts its conformation according to temperature to maintain microtubule networks in cells exposed low temperature (Delphin et al., 2012). Moreover, the recent production of pure tubulin isoforms has definitely established inherent isoform-specific differences in microtubule dynamics in vitro (Pamula et al., 2016; Ti et al., 2016; Vemu et al., 2016).
Genetic analyses in Drosophila melanogaster have provided the first unequivocal evidence for paralog-specific functions in vivo (Ludueña and Banerjee, 2008). The genome of D. melanogaster contains five alpha-tubulin genes, as well as five beta-tubulin genes (betaTub56D, betaTub60D, betaTub85D, betaTub97EF, and CG32396 designated here as betaTub65B). The most divergent beta-tubulin paralogs (betaTub85D and betaTub65B) are expressed exclusively in testis. While betaTub65B has not yet been studied, betaTub85D has been characterized in considerable detail. It is required in the germline for male meiotic divisions and sperm axoneme formation (Fuller et al., 1988; Kemphues et al., 1982). These processes still fail when betaTub60D, which is not normally expressed in the male germline, is expressed instead of betaTub85D (Hoyle and Raff, 1990). The normal expression of betaTub60D is also tissue-specific, but considerably more complex than that of the testis-specific betaTub85D. During embryogenesis betaTub60D expression starts in differentiating mesodermal cell types, but occurs also in chordotonal organs, imaginal discs and somatic cells of the adult gonads (Kimble et al., 1990; Rudolf et al., 2012). The zygotic expression of betaTub56D has also pronounced tissue-specificity, but at the start of embryogenesis the abundant maternal betaTub56D contribution is the only source of beta-tubulin (Buttgereit et al., 1996).
Here we report that betaTub97EF, an uncharacterized beta tubulin paralog, is upregulated at low temperature in specific cell types. Moreover, we demonstrate that it increases microtubule stability.
Results
betaTub97EF encodes a temperature-regulated beta-tubulin
Genome-wide expression profiling with Drosophila S2R+ cells over a range of different growth temperatures revealed betaTub97EF among the temperature-responsive genes. Compared to 25°C, the presumed optimal temperature, betaTub97EF transcript levels were increased at lower temperatures (11 and 14°C) and decreased at a higher temperature (30°C) in multiple independent experiments (Fig. 1A; data not shown). In another cell line, embryonic cells immortalized exploiting Act5c>Ras85DG12V as described recently (Simcox et al., 2008), betaTub97EF transcript levels were also twofold higher at 14°C compared to 30°C. In contrast to betaTub97EF, other tubulin genes were at most marginally affected by growth temperature (Fig. 1A). Independent analyses with quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed the microarray results (Fig. 1B,C). According to qRT-PCR, betaTub97EF transcripts amount to about 8% of the total beta-tubulin transcripts, while betaTub56D and betaTub60D contribute 46% each in S2R+ cells at 25°C (Fig. 1B). At 14°C, betaTub97EF transcripts are more than twofold higher (Fig. 1C).
To study temperature effects on protein abundance, we raised an antibody against beta-Tubulin 97EF using a C-terminal peptide for immunization. The C-terminal tails are the most divergent regions of the highly conserved beta-tubulins (Fig. S1). They have previously permitted production of paralog-specific antibodies (Kimble et al., 1989; Leiss et al., 1988). Immunoblotting experiments with extracts from bacteria expressing GST fusion proteins with extensions identical to the tails of the different beta-tubulin paralogs were used for initial antibody characterization (Fig. S2A,B). Immunoblotting with these extracts also demonstrated that the mouse monoclonal antibody E7 (Chu and Klymkowsky, 1989) detects with almost equal sensitivity all of the beta-tubulin paralogs, except beta-Tubulin 65B, a most divergent uncharacterized paralog with low testis-specific expression. The specificity of the newly generated antibodies for beta-Tubulin 97EF was further confirmed by immunoblotting and immunolabeling experiments using betaTub97EF mutants (see below). Quantitative immunoblotting indicated that the amount of beta-Tubulin 97EF in S2R+ cells is only about 2% of the total beta tubulin protein at 25°C (Fig. S1C). However, immunoblotting with extracts of S2R+ cells grown at different temperatures demonstrated that beta-Tubulin 97EF levels were temperature-dependent. Although slightly less pronounced than betaTub97EF transcripts, protein was significantly more abundant at low temperature (11 and 14°C) and reduced at high temperature (30°C) compared to 25°C (Fig. 1D,E). As expected (Radermacher et al., 2014), control immunoblotting with RL2, an antibody recognizing O-GlcNAc modified proteins, revealed the opposite temperature dependence. In conclusion, in cultured cells both mRNA and protein levels of betaTub97EF respond to temperature. Higher levels are present at low temperatures.
betaTub97EF expression occurs predominantly in the gut
Three of the five beta-tubulin paralogs, betaTub56D, betaTub60D and betaTub85D have been studied in considerable detail (Rudolf et al., 2012, and references given therein) but not betaTub97EF. Therefore, the pattern of betaTub97EF expression during development at 25°C was analyzed. By immunoblotting (Fig. 2A), beta-Tubulin 97EF could not be detected at the onset of embryonic development, consistent with RNA-Seq data (Graveley et al., 2011). Zygotic expression started between 6-9 hours after egg deposition (AED) and increased during the second half of embryogenesis. The specificity of the corresponding immunoblot signals (Fig. 2A) was confirmed with extracts of betaTub97EF mutant embryos (see below).
For comparison, immunoblotting with anti-beta-Tubulin 60D confirmed an expression program similar to beta-Tubulin 97EF with a slightly earlier onset and peak of zygotic expression (Fig. 2A). Moreover, anti-pan-beta-tubulin E7 revealed maximal levels of total beta-tubulin at the start of embryogenesis (Fig. 2A) when the abundant maternally contributed beta-Tubulin 56D is present (Rudolf et al., 2012). The monoclonal antibody DM1A (Blose et al., 1984) was used to detect alpha-tubulins (Fig. 2A). Based on its epitope characterization (Breitling and Little, 1986), DM1A presumably reacts with all or most of the Drosophila alpha-tubulins but the precise affinities for the different isoforms are not known.
Immunofluorescent labeling was performed to determine the spatial pattern of beta-Tubulin 97EF expression during embryogenesis (Fig. 2B). beta-Tubulin 97EF was not detected until stage 12 when weak specific signals were observed in the developing hindgut. Subsequently signal intensities increased in the hindgut. Expression was also detected in foregut and posterior spiracles, as well as more weakly in the midgut (Fig. 2B). The predominantly gut-specific expression pattern of beta-Tubulin 97EF is clearly distinct from that of beta-Tubulin 60D with its primarily muscle-specific expression pattern (Kimble et al., 1989; Leiss et al., 1988), and data not shown). The maternally contributed, abundant beta-Tubulin 56D is ubiquitously and uniformly distributed during embryogenesis, as indicated by E7 staining (data not shown).
To characterize anti-beta-Tubulin 97EF staining in further detail, the region with the embryonic hindgut was analyzed at high magnification (Fig. 2C). This revealed beta-Tubulin 97EF in the epithelial cells of the hindgut, but not in the surrounding visceral muscles. Anti-beta-Tubulin 60D resulted in the opposite pattern (data not shown). Within the hindgut epithelium, beta-Tubulin 97EF was most abundant in the so-called boundary cells (Fig. 2C), a row of single cell width extending along both sides of the hindgut tube between the ventral and dorsal compartment (Iwaki and Lengyel, 2002).
At maximal resolution, near perfect spatial overlap of the signals obtained by double labeling with anti-beta-Tubulin 97EF and anti-alpha-tubulin was observed (Fig. 2D). For these high resolution analyses, we used released larval hemocytes which spread out flat on coverslips, providing superior optical conditions. Although beta-Tubulin 97EF was colocalized with alpha-tubulin, the spatial intensity distributions of the signals obtained by double labeling with the two antibodies were not identical. Accordingly, beta-Tubulin 97EF might be enriched in cortical microtubules, but alternative explanations are not excluded (see discussion).
Immunolabeling revealed that beta-Tubulin 97EF is also present in the larval and adult gut (Fig. S3B,C). Signals in enterocytes were rather uniform, i.e., not as in the embryonic hindgut where boundary cells have higher levels. beta-Tubulin 97EF was not only detected in the gut. Specific signals, although weaker, were observed in wing imaginal discs, larval hemocytes and ovarial follicle cells (see below and data not shown).
We conclude that betaTub97EF expression occurs in a pattern distinct from that of other beta-tubulin paralogs. In situ hybridization and a betaTub97EF>GFP reporter allele suggested that tissue and cell-type specificity of betaTub97EF expression is transcriptionally controlled (data not shown).
betaTub97EF upregulation by low temperature is tissue-specific
To address the physiological role of betaTub97EF, we characterized mutant alleles (Fig. 3A). Three Minos transposon insertions in the betaTub97EF region were analyzed (Fig. 3A). PCR assays confirmed the annotated insertion positions. The insertion Mi{MIC}betaTub97EFMI06334 within the first intron (betaTub97EFMiMIC in the following) introduces a splice acceptor site followed by translational stops in all three reading frames. By conversion of this insertion (Venken et al., 2011), a derivative with an extra EGFP-encoding exon within this first intron was obtained (betaTub97EFEGFP). The predicted protein product is expected to be non-functional because the EGFP insertion disrupts the tubulin core domain. Indeed, beta-Tubulin97EFEGFP was not incorporated into microtubules according to fluorescence microscopy.
By qRT-PCR (Fig. 3B) and immunoblotting (Fig. 3C), we analyzed how betaTub97EF expression was affected by the transposon insertions. While two, Mi{ET1}betaTub97EFMB01877 and Mi{ET1}betaTub97EFMB03812, reduced expression, betaTub97EFMiMIC appeared to abolish it completely (Fig. 3B,C). Residual beta-Tubulin 97EF, potentially expressed from the betaTub97EFMiMIC allele, cannot be more than 3% of the normal level, because stronger expression would have been detectable (Fig. 3C). In case of betaTub97EFEGFP, immunoblotting confirmed the expected expression of a larger beta-Tubulin 97EF variant with an internal EGFP domain (Fig. 3C). As anti-beta-Tubulin 97EF also revealed a weak band at the position of normal beta-Tubulin 97EF, rare skipping of the extra EGFP exon present in the betaTub97EFEGFP allele cannot be excluded and might allow expression of normal beta-Tubulin 97EF up to 10% of wild-type levels. Alternatively, the weak band might represent one of the obvious beta-Tubulin 97EFEGFP degradation products, rather than residual normal beta-Tubulin 97EF.
Unexpectedly given the conservation of the betaTub97EF ortholog during an estimated 300 million years of insect evolution (Findeisen et al., 2014), betaTub97EF function was found to be dispensable for viability and fertility under standard laboratory conditions. betaTub97EFMiMIC could be propagated as a homozygous stock. Compared to w1, fertility was 25% lower in both sexes in flies with betaTub97EFMiMIC over a deficiency deleting betaTub97EF (Df(3R)BSC460).
Since betaTub97EF expression is especially high in boundary cells of the embryonic hindgut, we analyzed whether these cells are present in betaTub97EFMiMIC mutants. Labeling of Crumbs, an apical membrane protein expressed at very high levels in boundary cells (Kumichel and Knust, 2014), demonstrated that these cells are present in betaTub97EFMiMIC mutants (Fig. 3D). To compare hindgut morphology in betaTub97EFMiMIC mutants and control embryos with ultrastructural resolution, we applied focused ion beam scanning electron microscopy (FIBSEM). While this failed to expose abnormalities in betaTub97EFMiMIC mutants, our three-dimensional reconstructions revealed that boundary cells differentiate striking undulae propagating longitudinally from cell to cell along the hindgut epithelial tube (Fig. S4), rather than prominent microvilli as suggested previously based on two-dimensional EM analyses (Iwaki and Lengyel, 2002; Kumichel and Knust, 2014; Soplop et al., 2012).
The hindgut is one of the organs that develops left/right asymmetry during development (Coutelis et al., 2014). betaTub97EFMiMIC mutants (n = 100) were found to have normal left/right asymmetry in larval and adult hindgut. We also addressed whether absence of beta-Tubulin 97EF from the gut might increase sensitivity to hyperosmotic diet. Therefore, survival to the adult stage after larval development in high salt food was analyzed. However, betaTub97EFMiMIC mutants were not more salt-sensitive than w1 control (data not shown).
Our phenotypic analyses suggested that betaTub97EF does not provide crucial functions at temperatures close to the optimum of 25°C. However, since betaTub97EF expression in S2R+ cells is increased at low temperature (Fig. 1), this gene might be of a greater importance during development and life at sub-optimal temperatures. First we analyzed whether regulation of betaTub97EF expression by temperature occurs also in embryos. After egg collection at 25°C, aliquots were shifted at 3-6 hours AED to either 14, 25 or 30°C. Once embryos had reached stage 16 (peak of betaTub97EF expression), total RNA was analyzed by qRT-PCR. Among tubulin paralogs, betaTub97EF is unique with regard to the presence of two mutually exclusive exons (4B and 4C) within the region coding for the conserved tubulin protein core (Fig. 3A) (Findeisen et al., 2014). These two mutually exclusive exons appear to be characteristic for dipterans (Findeisen et al., 2014). In D. melanogaster they share 77% identity at the amino acid sequence level (Fig. S1). Based on splice junction counts (Graveley et al., 2011), the 4B variant is about 15 fold more abundant than the 4C variant throughout development. Our qRT-PCR analyses were done with two distinct primer pairs designed to detect specifically one or the other isoform. While the minor 4C transcripts were found to be downregulated, the major 4B transcripts were up-regulated at lower temperature (11°C) (Fig. 4A).
Aliquots of the same embryos were also analyzed by immunoblotting (Fig. 4B). This confirmed that beta-Tubulin 97EF protein was up-regulated after development at low temperature. On average, beta-Tubulin 97EF levels were fourfold higher at 14°C compared to 30°C (Fig. 4B). Although with variation in extent, higher beta-Tubulin 97EF levels at the lower temperature were consistently observed in each of three experiments. In contrast, signals obtained by immunoblotting with anti-beta-Tubulin 60D and anti-alpha-tubulin were not affected by temperature (Fig. 4B), indicating that induction by low temperature is specific for the betaTub97EF paralog.
To address whether the upregulation of beta-Tubulin 97EF at low temperature occurs globally or in a tissue-specific manner we performed quantitative immunofluorescent labeling. To achieve maximal accuracy and minimize problems caused by potential fixation and staining artefacts, these experiments were performed by combining four different types of stage 16 embryos before fixation and staining in a pool (Fig. 4C). Two of these embryo types were processed after development at 14°C (Fig. 4C, blue). To identify these “cold” embryos during the microscopic analyses, they were collected from strains expressing red fluorescent centromere protein Cenp-C (g-tdTomato-CenpC) (Fig. 4C, red stars). In contrast, the two other types, the “warm” embryos, processed after development at a higher temperature (25°C or 30°C), did not have this marker transgene. Based on red fluorescent centromere signals (as illustrated with the brain lobe regions in Fig. 4C) “cold” and “warm” embryos were identified. Moreover, in order to quantify the level of non-specific signals resulting from anti-beta-Tubulin 97EF labeling, both the “cold” and the “warm” embryos were a mixture. Half were betaTub97EFMiMIC mutants and the other half had two wild-type alleles of betaTub97EF (Fig. 4C, + and -, respectively). Green fluorescent anti-beta-Tubulin 97EF labeling revealed the betaTub97EF genotype during the microscopic analyses. In addition, anti-alpha-tubulin labeling was visualized using a far-red fluorophore.
Quantification of the specific, background-corrected anti-beta-Tubulin 97EF signals indicated that beta-Tubulin 97EF levels were 1.7 fold higher in the embryonic hindgut at 14°C compared to 25°C, and 2 fold higher when compared to 30°C (Fig. 4D,E). Tissue-specific beta-Tubulin 97EF upregulation in the hindgut at low temperature was also observed when g-tdTomato-CenpC was used for marking the “warm” instead of the “cold” embryos. In contrast, anti-alpha-tubulin signals were not affected by temperature (Fig. 4D,E). Analogous quantification of signals within epidermis and somatic muscles did not reveal any temperature effects, neither on beta-Tubulin 97EF nor on alpha-tubulin signals (data not shown).
Although low temperature induces higher beta-Tubulin 97EF expression in the hindgut, this organ appeared to be morphologically normal in betaTub97EFMiMIC mutants even after embryonic development at 14°C or 12°C (data not shown). Despite normal morphology, certain cells or tissues might not be fully functional in betaTub97EFMiMIC mutants. Therefore we analyzed temperature sensitivity of development to the larval and adult stages. Embryos were collected at 25°C and aliquots were shifted after gastrulation, but before the onset of betaTub97EF expression, to temperatures within the range of 10 to 34°C (Fig. 4F). The rate of larval hatching from eggs (Fig. 4G) as well as the rate of development to the adult stage was determined (Fig. 4H). At 10°C, no larvae were observed to hatch, neither from control nor from betaTub97EF mutant eggs. Similarly, only very few larvae hatched at 11°C and 12°C (Fig. 4G). Interestingly, compared to control, 25% fewer betaTub97EFMiMIC mutants completed embryogenesis successfully at 14°C (Fig. 4G). This temperature of 14°C is the lower limit for completion of the entire life cycle for standard D. melanogaster laboratory strains. Therefore, betaTub97EF function appears to be important at the lower end of the temperature range compatible with the complete life cycle. In contrast at 30°C, the upper end of this compatible range, there were no significant survival differences between betaTub97EFMiMIC mutants and control, as well as at the optimum temperature of 25°C (Fig. 4G). Only at more drastically elevated temperatures, embryogenesis of betaTub97EF mutants was less successful than in controls (Fig. 4G). When analyzing development up to the adult stage, betaTub97EF mutants were found to be significantly weaker than control at temperatures below as well as above the optimum (Fig. 4H). By immunoblotting with total larval extracts, we were actually unable to detect beta-Tubulin 97EF upregulation at low temperature at this developmental stage (data not shown). In conclusion, within the temperature range where the full D. melanogaster life cycle can be completed with a normal genotype, the importance of betaTub97EF function for successful embryogenesis increases towards the lower limit, supporting the notion that betaTub97EF upregulation at low temperature contributes to acclimation.
beta-Tubulin 97EF generates stable microtubules
Functional redundancy of co-expressed beta-tubulin paralogs might explain the mild phenotypic consequences of betaTub97EF null mutations. In betaTub97EF mutant embryos, the maternally provided abundant beta-Tubulin 56D (Buttgereit et al., 1991) is present at least initially. To address potential functional redundancy, we first characterized betaTub56D mutant alleles. We studied two transposon insertions, betaTub56DNP0949 and betaTub56DEY12330, that appeared likely to be detrimental for betaTub56D function (Fig. 5A). Both insertions were found to be homozygous lethal, as well as over a deficiency (Df(2R)BSC782) that eliminates 5’ upstream sequences including the promoter region and also the first betaTub56D exon (Fig. 5A). Moreover, the insertions failed to complement each other. Finally, the developmental lethality associated with betaTub56DNP0949/Df(2R)BSC782 was completely suppressed by UASt-betaTub56D expression driven by alphaTub84BP-GAL4 (Fig. 5A). Therefore, betaTub56DNP0949 and betaTub56DEY12330 are loss of function alleles of betaTub56D.
The lethality associated with betaTub56DNP0949/Df(2R)BSC782 occurred after embryogenesis during the first larval instar (Fig. 5A), suggesting that the maternal betaTub56D contribution is sufficient for successful completion of embryogenesis. Indeed, the abundant maternal betaTub56D contribution was found to be stable throughout embryogenesis, as revealed by immunolabeling experiments using Df(2R)BSC782 (data not shown). This maternal betaTub56D contribution might explain why hindgut development appeared to be normal even when zygotic function of both betaTub97EF and betaTub56D were missing during embryogenesis (data not shown).
Paralog retention after gene duplication during evolution usually depends on functional specialization at the level of expression and/or protein function. To address protein specialization, we compared the betaTub56D mutant rescue potential of the different beta-tubulin paralogs (Fig. 5A). For paralog expression in betaTub56D mutants (betaTub56DNP0949/Df(2R)BSC782), we always used the same GAL4 driver (alphaTub84BP-GAL4) in combination with UASt transgenes that differed only within the coding region. The coding region was that of the beta-tubulin paralogs 56D, 60D, 65B, 85D, and 97EF with either exon 4B or 4C. As described above, UASt-betaTub56D expression resulted in complete rescue of betaTub56DNP0949/Df(2R)BSC782 first instar larval lethality (Fig. 5A). In contrast, UASt-betaTub97EF(4B) and UASt-betaTub97EF(4C) provided only limited rescue. Lethality still occurred, but during the second larval instar rather than already during the first (Fig. 5A). While such a partial rescue was also obtained with UASt-betaTub65B, the others (UASt-betaTub60D and UASt-betaTub85D) did not appear to cause rescue (Fig. 5A).
Additional experiments with the UASt transgenes revealed that ectopic expression of some beta-tubulin paralogs is highly toxic in an otherwise normal background. When the UASt transgenes were expressed with ey-GAL4 during eye development for example (Fig. 5B), the beta-Tubulin 97EF isoforms were more toxic than all the other beta-tubulin paralogs. While the variant with the 4C exon was lethal, the 4B version resulted in small deformed and rough eyes, indicating that the two beta-Tubulin 97EF isoforms are functionally distinct.
When mat-4-GAL-VP16 was used to drive UASp-betaTub97EF(4B) during oogenesis, female sterility was observed and the dumping of nurse cell cytoplasm into the oocyte that normally occurs during stage 10B was inhibited (Fig. 5C,D). Such a dumpless egg chamber phenotype was not obtained with UASp-betaTub85D or UASp-betaTub60D (Fig. 5D). Immunolabeling confirmed that ectopic expression of both the toxic beta-Tubulin 97EF and the non-toxic beta-Tubulin 60D occurred as expected in the germline starting during stage 2 (Fig. S5). During wild-type oogenesis, both beta-Tubulin 97EF and beta-Tubulin 60D are normally expressed in somatic follicle cells but not in the germline (Fig. S5).
The C-terminal tails of tubulin paralogs are the most divergent regions and different types of posttranslational modifications occur predominantly in this region, potentially generating a tubulin code that might direct interactions with specific sets of MAPs and motor proteins. To evaluate whether the functional difference between beta-Tubulin 97EF(4B) and beta-Tubulin 56D reflects sequence changes within the core or the C-terminal tail, we performed betaTub56D mutant rescue experiments with UASt transgene encoding tail-swopped variants. This assay indicated that the functional difference between beta-Tubulin 97EF(4B) and beta-Tubulin 56D reflects alterations within both the core and the C-terminal tail regions (Fig. 5A). We conclude that betaTub97EF is not just expressed in a specific pattern but that it also codes for a beta-tubulin with unique functions as a result of sequence changes within both the tubulin core and the C-terminal tail region.
For further analysis of the functional differences between beta-Tubulin 97EF and beta-Tubulin 56D, we studied larval hemocytes. Since S2R+ cells have an expression profile most similar to hemocytes (Cherbas et al., 2011), it appeared likely that larval hemocytes express betaTub97EF in a temperature-regulated manner. Indeed, immunolabeling confirmed that larval hemocytes do not only express betaTub56D but also betaTub97EF (Figs 2D, 6A). Moreover, the beta-Tubulin 97EF level was significantly increased at 14°C compared to 29°C (Fig. 6B). By exploiting larval hemocytes from betaTub56D mutants in combination with expression of UASt tubulin transgenes, we were able to alter the relative abundance of beta-Tubulin 97EF and beta-Tubulin 56D even at the optimal temperature of 25°C (Fig. 6A). Moreover, coexpression of UASp-EB1-GFP allowed quantitative analyses of some aspects of microtubule dynamics in vivo. EB1-GFP accumulates on the plus ends of growing microtubules (Mimori-Kiyosue et al., 2000). Therefore, we used hemocytes from second instar larvae with mutations in betaTub56D (betaTub56DNP0949/Df(2R)BSC782) and alphaTub84BP-GAL4 driven expression of UASt-betaTub97EF(4B) or UASt-betaTub56D and also UASp-EB1-GFP in some of the experiments. In the following, these hemocytes will be designated as R-97EF and R-56D (rescued by the 97EF and 56D paralog, respectively). In addition, we also analyzed hemocytes from siblings (S-97EF and S-56D, respectively) which expressed the UAS transgenes in balanced Df(2R)BSC782 larvae with a functional endogenous betaTub56D copy on the balancer.
After immunolabeling, we quantified the signals obtained with anti-beta-Tubulin 97EF, DM1A (anti-alpha-tubulin) and E7 (anti-beta-tubulin) in the hemocytes (Fig. 6A). We point out that these signals primarily reflect the polymerized tubulin pool rather than the total cellular pool. Fixation seems to result in a loss of soluble tubulin, as suggested by the comparison of anti-tubulin immunolabeling signal intensities after brief pretreatment with either microtubule inhibitors (nocodazole, vinblastine) or buffer, respectively (Bossing et al., 2012; Schulman et al., 2013); see also Fig. 7). In addition, while non-specific background intensity resulting with anti-beta-Tubulin 97EF, as evident in betaTub97EF null mutant hemocytes, was subtracted from signal intensities obtained in the other genotypes, the extent of non-specific background resulting with DM1A (anti-alpha-tubulin) and E7 (anti-beta-tubulin) could not be assessed and subtracted. Moreover, non-linearity of detection might vary between different antibodies. Despite these limitations our signal quantification clearly revealed that expression of UASt-betaTub97EF(4B) and UASt-betaTub56D, respectively, has distinct consequences in larval hemocytes. UASt-betaTub97EF(4B) expression resulted in a pronounced increase of not only the signals obtained with anti-beta-Tubulin 97EF and E7, but also of those obtained with anti-alpha-tubulin (Fig. 6A, R-97EF and S-97EF). The anti-tubulin signals were particularly prominent in microtubule bundles along the cortex. In contrast, UASt-betaTub56D expression resulted in a more limited increase in signal intensities (Fig. 6A, R-56D and S-56D). Therefore, beta-Tubulin 97EF appears to stabilize microtubules. The posttranscriptional feedback preventing free tubulin heterodimer excess (Cleveland, 1988) likely contributes to the positive effect of UASt-betaTub97EF(4B) expression on anti-alpha-tubulin signal intensity.
For additional comparison of the effects resulting from UASt-betaTub97EF(4B) and UASt-betaTub56D expression, we analyzed EB1-GFP comet movements. In R-56D and S-56D hemocytes, moving EB1-GFP comets were readily observed (Fig. 6C, Suppl Movie 1), similar as previously described in normal control hemocytes (Leśniewska et al., 2014). In contrast, EB1-GFP comets were far less evident in R-97EF and S-97EF hemocytes (Fig. 6C, Suppl Movie 2). In these cells, the mobile comets were partially masked by stably persisting EB1-GFP signals all along the microtubule network and most prominently at the cell cortex. Newly appearing EB1-GFP comets advanced mostly along the persisting bundles. Quantification of the dynamics of EB1-GFP comets did not reveal statistically significant differences in the effects of UASt-betaTub97EF(4B) and UASt-betaTub56D expression on the speed of EB1-GFP comet migration (Fig. 6C). These findings suggested that beta-Tubulin 97EF increases the amount of microtubules, but not by stimulating the microtubule growth rate. Thus it presumably decreases the catastrophe frequency and/or it increases the rescue frequency.
To further confirm that increased levels of beta-Tubulin 97EF stabilize microtubules, we analyzed their colcemid sensitivity. Colcemid inhibits microtubule growth and depolymerizes microtubules at higher concentrations (Ravelli et al., 2004). Colcemid was found to have a more pronounced effect on hemocytes expressing UASt-betaTub56D compared to those expressing UASt-betaTub97EF(4B) (Fig. 6D). In R-56D hemocytes, anti-alpha-tubulin signals were drastically lower after one hour incubation in the presence of colcemid. In contrast, signals were still high in R-97EF hemocytes even after 2 hours of incubation although in a more patchy distribution. Analogous analyses after expression of beta-Tubulin 56D and 97EF with swopped C-terminal tails, indicated that the microtubule stabilizing effect is mediated by the beta-Tubulin 97EF core and not by its C-terminal tail (Fig. 6D). Our results support the notion that beta-Tubulin 97EF stabilizes microtubules.
The analysis of microtubules in hemocytes indicated that microtubules containing beta-Tubulin 97EF are more stable than those built primarily from the major embryonic maternally provided beta-Tubulin 56D. Accordingly, the hindgut microtubules of betaTub97EF mutants (containing only maternally derived beta-Tubulin 56D) are predicted to be less stable than those in wild-type control embryos (containing beta-Tubulin 56D and beta-Tubulin 97EF). To address this prediction, we performed microtubule depolymerization experiments with embryos with or without betaTub97EF function. For these experiments, we used vinblastine because it depolymerized microtubules in embryos more effectively than colcemid (data not shown). To enforce equal conditions during vinblastine treatment as well as during subsequent fixation and immunolabeling, embryos with and without betaTub97EF function were combined into a pool before treatment onset. To identify the different genotypes during the microscopic analysis eventually, the embryos with betaTub97EF gene function had the g-tdTomato-CenpC marker gene, resulting in red fluorescent centromere signals. In contrast, the betaTub97EF mutants did not have this marker transgene. After vinblastine or mock treatment, embryos were stained with anti-alpha-tubulin and signal intensities were quantified. The quantification revealed that in the hindgut, vinblastine caused a significant reduction in the level of anti-alpha-tubulin signals but only in embryos lacking betaTub97EF function (Fig. 7). In contrast, in the tissues surrounding the hindgut and in the body muscles, vinblastine reduced the level of anti-alpha-tubulin signals to a comparable extent in embryos with and without betaTub97EF function (Fig. 7). These results indicate that microtubules in normal hindgut, where betaTub97EF expression is high, are more stable than in tissues, where betaTub97EF is at most marginally expressed. Moreover, this higher microtubule stability in the hindgut depends on betaTub97EF expression, as it is lost in betaTub97EF mutants.
Discussion
The molecular mechanisms allowing acclimation to ambient temperature fluctuations are poorly understood. Here we have identified betaTub97EF as a temperature responsive gene in Drosophila melanogaster. At low temperature, betaTub97EF expression is increased. However, at the optimal temperature, as well as at lower temperatures, betaTub97EF is not expressed ubiquitously but restricted in a range of tissues including gut epithelial cells and hemocytes. Mutations that eliminate betaTub97EF function have a surprisingly mild effect, presumably reflecting functional redundancy with other co-expressed beta-tubulin paralogs. But without beta-Tubulin 97EF, microtubules are less stable. Conversely, microtubules are stabilized by increased betaTub97EF expression. Moreover, within the well-tolerated temperature range, embryogenesis of betaTub97EF mutants is more cold-than heat-sensitive. Therefore, upregulation of betaTub97EF at low temperature might contribute to acclimation by stabilizing microtubules.
A detailed understanding of the betaTub97EF contribution to the success of embryogenesis specifically at the lower end of the temperature range compatible with completion of the entire life cycle will require further work. Lack of beta-Tubulin 97EF in the gut epithelia might impair yolk utilization during late embryogenesis at low temperature. It might also affect hemocyte functions (Wood and Martin, 2017). Hemocytes are important for innate immunity, which is unlikely to be relevant for successful embryogenesis. However, they also provide functions essential for embryogenesis. They scavenge apoptotic corpses resulting from the abundant developmentally programmed cell death. Moreover, they are crucial for secretion and remodeling of much of the extracellular matrix in embryos.
Microtubules are ubiquitous and temperature change will affect them in all cells. Irrespective of temperature change, microtubule properties are carefully controlled in animal cells, not only by alterations in tubulin isoform composition, but also by myriads of interacting factors. Thereby their characteristics in different cell types and intercellular subsets are modified to serve a wide range of functions. Acclimation of these diverse microtubule systems to temperature change is therefore likely demanding and impossible to achieve simply and exclusively by global upregulation of a single tubulin paralog. Presumably, tissue-specific upregulation of betaTub97EF is one of many mechanisms for microtubule acclimation in D. melanogaster.
Beyond acclimation to low temperature, betaTub97EF expression occurs also at optimal and elevated growth temperatures. betaTub97EF is one of the five beta-tubulin paralogs present in the genome of D. melanogaster, which includes five alpha-tubulin paralogs. The human genome hosts almost twice as many alpha- and beta-tubulin genes (9 and 8, respectively) and genetic analysis during the last 10 years have uncovered an increasing number of disease-associated mutations primarily in the context of neurological disease and cancer (Bahi-Buisson et al., 2014; Ludueña and Banerjee, 2008; Wang et al., 2017). Characterization of these genes and associated phenotypes, also in mouse models, has fully confirmed the conclusions from pioneering analyses of tubulin paralog functions in D. melanogaster. Tubulin paralogs have distinct but often partially overlapping patterns of expression and functions. The understanding of how amino acid sequence alterations affect tubulin function has progressed slowly. Production of pure recombinant tubulin isoforms in quantities sufficient for studies of microtubule polymerization and dynamics in vitro has been achieved only very recently (Pamula et al., 2016; Sirajuddin et al., 2014; Ti et al., 2016; Valenstein and Roll-Mecak, 2016; Vemu et al., 2016; Widlund et al., 2012). As the microtubule interactome in cells is highly complex (Hughes et al., 2008; Yu et al., 2016) phenotypic analyses in vivo will remain important as well.
Here we have used a series of transgenes for GAL4-regulated expression of all the D. melanogaster beta-tubulin paralogs. As the transgenes are identical except for the coding sequences, differential effects most likely reflect functional specialization at the protein level. Expression with ey-GAL4 (and additional drivers including en-GAL4 and da-GAL4, data not shown) indicated that excess betaTub97EF is more toxic than that of the other beta-tubulin paralogs. Moreover, betaTub97EF-4C is more detrimental than betaTub97EF-4B. These two betaTub97EF isoforms, resulting from mutually exclusive splicing of the fourth exon, differ at 13 positions within an internal segment of 40 amino acids, but their N- and C-terminal regions are identical. Sequence divergence between tubulin paralogs is generally most extensive within the tails, in particular C-terminally where also several distinct post-translational modifications occur (Gadadhar et al., 2017; Janke, 2014; Yu et al., 2015). Therefore, the C-terminal tails (CTTs) are thought to make important paralog-specific contributions, as fully confirmed recently (Sirajuddin et al., 2014). Our comparison of the 97EF and 56D beta-tubulins also revealed that CCTs contribute to paralog-specific function. While 56D with an 97EF-CTT behaved like normal 56D, 97EF with an 56D-CTT had a toxicity intermediate between 97EF and 56D, suggesting that the beta-Tubulin 97EF core and tail cooperate, as demonstrated most elegantly with the 56D and 85D paralogs (Popodi et al., 2008). Beyond CTT contributions, differences within the tubulin core were found to have more profound effects. The core is responsible for the characteristic microtubule stabilizing effect of beta-Tubulin 97EF in larval hemocytes. Similarly, a study with chimeric tail-swapped human beta-tubulins IIB and III concluded that differences between the cores were largely responsible for the different microtubule dynamics observed in vitro (Pamula et al., 2016).
Our results indicate that beta-Tubulin 97EF expression does not affect the microtubule growth rate but it results in microtubules that are less prone to destabilization by colcemid and vinblastine. As beta-Tubulin 97EF appears to be enriched in cortical microtubules, it might confer resistance against cortical factors that stimulate catastrophe. Cortical enrichment was apparent after double labeling of larval hemocytes with anti-alpha–tubulin and anti-beta-Tubulin 97EF. However, locally distinct epitope masking effects, for example by CTT-associated proteins or modifications, are clearly not excluded. Judging from the pattern of microtubules induced by beta-Tubulin 97EF overexpression in hemocytes, this tubulin might also facilitate microtubule bundling (Molodtsov et al., 2016). Interestingly, while ectopic betaTub60D expression in the germline did not affect oogenesis, betaTub97EF specifically blocked dumping of nurse cell cytoplasm into the oocyte during mid-oogenesis, i.e. long after the onset of ectopic expression. Dumping is critically dependent on actin and myosin. Microtubule inhibitors, which clearly inhibit some aspects of transport from nurse cells to oocyte, do not block the dumping process (Gutzeit, 1986; Lu et al., 2016; Roth and Lynch, 2009; Wheatley et al., 1995, Huelsmann, 2013 #7420). An excess of cortical microtubule bundles caused by ectopic beta-Tubulin 97EF might therefore inhibit dumping by interference with normal myosin-mediated contraction of the cortical actin network in nurse cells. Evidently, further analyses will be required to evaluate our speculative proposals. It is clear, however, that beta-Tubulin 97EF has interesting properties that are remarkably distinct from those of other D. melanogaster paralogs.
Materials and methods
Drosophila strains
Several stocks were obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN, USA) and the Kyoto Stock Center (Kyoto Institute of Technology, Kyoto, JP): betaTub97EFMiMIC (#41519) (Venken et al., 2011), betaTub97EFMBO1877 (# 24452) and betaTub97EFMBO3812 (#24275) (Metaxakis et al., 2005), betaTub56DEY12300 (#20734) (Bellen et al., 2004); Df(2R)BSC782 (#27354) and Df(3R)BSC460 (#24964) (Cook et al., 2012), betaTub56DNP0949 (#103830) (Hayashi et al., 2002), betaTub97EFEGFP was generated as described in further detail (Supplementary Information). Two strains with UASt-Tub xy transgenes were obtained from FlyORF (University of Zurich, Zurich, Switzerland): M{UAS-betaTub85D.ORF}ZH-86Fb (F001298) and M{UAS-betaTub97EF(4B).ORF}ZH-86Fb (F001587) (Bischof et al., 2013). Additional UASt-Tub lines (M{UAS-betaTub56D.ORF}ZH-86Fb, M{UAS-betaTub60D.ORF}ZH-86Fb, M{UAS-betaTub65B.ORF}ZH-86Fb, M{UAS-betaTub97EF(4C).ORF}ZH-86Fb, M{UAS-betaTub56D-97EFtail.ORF}ZH-86Fb M{UAS-betaTub97EF(4B)-56Dtail.ORF}ZH-86Fb) as well as the UASp-Tub lines M{UASp-betaTub97EF(4B).ORF}ZH-86Fb and M{UASp-betaTub60D.ORF}ZH-86Fb were generated as described in further detail (Supplementary Information). UASp-EB1-GFP (Jankovics and Brunner, 2006) was kindly provided by D. Brunner (University of Zurich, Zurich, Switzerland). The GAL4 driver and marker lines have been described previously: ey-GAL4 (5/8) (Hazelett et al., 1998), mat 4-GAL-VP16(V2H) (Hacker and Perrimon, 1998), alphaTub84BP-GAL4 (LL7) (#5138) (Lee and Luo, 1999) and gi2xtdTomato-Cenp-C II.3 (Althoff et al., 2012).
For analysis of the extent of betaTub56D mutant development supported by other beta-tubulin paralogs, the following crosses were set up. Virgins Df(2R)BSC782/CyO, Dfd-YFP; UASt-betaTubXY with XY = 56D, 60D, 65B, 85D, 97EF(4C) or 97EF(4C) were crossed with males w*; betaTub56DNP0949/CyO, Dfd-GMR-nvYFP; alphaTub84BP-GAL4/TM6B, Hu, Dfd-YFP. Eggs were collected from these crosses on either apple agar plates or in tubes with standard fly food. Collection plates were inspected at intervals under a fluorescence stereomicroscope allowing identification of larvae without the Dfd-YFP balancer chromosomes.
As control strain, w was used. betaTub97EFMiMIC was backcrossed for four generations to w before comparison of the temperature sensitivity of development.
The rate of larval hatching from eggs was determined as described previously (Radermacher et al., 2014). Eggs were collected during 60 minutes on apple agar plates at 25°C and aged at this temperature for 5 hours. The collection plates with the eggs were then divided into sectors. The eggs on one of the sectors were fixed immediately with methanol and stained with a DNA stain to determine the fraction of unfertilized eggs. The other sectors were shifted to different temperatures and the fraction of hatched and unhatched eggs was determined eventually with the help of a stereomicroscope. The hatch rate was calculated as the ratio of the number of hatched eggs to the total number of eggs deposited on the sector minus the number of unfertilized eggs. For the analysis of the rate of development to the adult stage, eggs were also collected during 60 minutes at 25°C followed by ageing at this temperature for 5 hours. Sectors of the collection plates with eggs that had been counted were then separated into bottles containing standard fly food and shifted to different temperatures. The number of eclosed flies was determined eventually. The eclosion rate was calculated as a ratio between the number of eclosed flies to the total number of eggs minus the number of unfertilized embryos.
Transcript analyses
The analysis of temperature effects on the transcriptome of cultured cells will be described in detail elsewhere. Data shown here was obtained with S2R+ cells which were cultured as described previously (Radermacher et al., 2014). Aliquots were shifted 24 hours after plating at 25°C to different temperatures (11, 14, 25 and 30°C). 24 hours later RNA was extracted, followed by probe synthesis and microarray hybridization (Drosophila gene expression 4x44K microarrays, G2519F-021791, Agilent Technologies). Three independent replicas were analyzed and after normalization of signal intensities across replicates average hybridization signals were determined. In contrast to all other tubulin paralogs, signals for the testis-specific betaTub85D paralog were too low to be included in the S2R+ transcriptome analysis (Fig. 1A).
For qRT-PCR analysis, S2R+ cells were treated in the same way as for the microarray experiments. For qRT-PCR analysis with w1 embryos, eggs were collected at 25°C for 3 hours. After additional ageing at 25°C for 3 hours, they were shifted to 14, 25 and 30°C for 40, 10 or 8 hours, respectively. As a result, embryos were all at stage 16 of embryogenesis irrespective of the applied temperature regime. An aliquot of each sample was fixed for controlling the developmental stage after DNA staining by microscopy. Further details concerning the qRT-PCR analyses, including primer sequences, are described in Supplementary Information.
Antibodies
Detailed information on all antibodies and dilutions applied during immunoblotting and immunolabeling is given in the Supplementary Information. The rabbit antibodies against beta-Tubulin 97EF were generated with a synthetic peptide corresponding to last 17 amino acids (AEQEGYESEVLQNGNGE) coupled to KHL (Moravian Biotechnology, Brno, Czech Republic). Antibodies were affinity purified. Rabbit antibodies against beta-Tubulin 60D (Leiss et al., 1988) were kindly provided by R. Renkawitz-Pohl (University of Marburg, Marburg, Germany).
Immunoblotting
Total extracts from cells, embryos, larvae and adults were prepared as described in further detail in the Supplementary Information and resolved by gel electrophoresis. Embryos and larvae aged at different temperatures were processed with solutions that had been pre-equilibrated to the temperatures used for aging. For probing samples with different anti-tubulin antibodies, separate gels and immunoblots were used for each antibody in order to avoid potential masking effects that might arise during serial probing of a single immunoblot. Immunoblot signals were developed using chemiluminescence (Amersham ECL, GE Health Care Life Sciences) and captured using a CCD camera. For quantification of immunoblot signals a dilution series of a reference extract was resolved along with the test samples. Quantification of signals by densitometry was done with ImageJ as described previously (Radermacher et al., 2014).
Microscopic analyses
Embryos were immunolabeled as described before (Radermacher et al., 2014). For vinblastine treatment, gi2xtdTomato-Cenp-C II.3 and betaTub97EFMiMIC embryos were collected and aged to 11-13 hours AED. Pooled embryos were dechorionated with 3% sodium hypochlorite, washed extensively with 0.7% NaCl, 0.07% Triton X-100 and transferred into an Eppendorf tube. 1.5 ml of a 1:1 mixture of heptane and Schneider’s medium were added. Moreover, 3 μl of a vinblastine (Sigma, V1377) stock solution (5 mM in methanol) or 3 μl methanol in case of control aliquots were added. The Eppendorf tubes were slowly rotated end over end for 20 minutes at room temperature. Thereafter, embryos were fixed with methanol.
Preparation and immunolabeling of organs from larvae and adults was performed using standard procedures as described in further detail in Supplementary Information.
Hemocytes were isolated as previously described (Sampson and Williams, 2012) using second instar larvae (R-56D, S-56D, R-97EF, S-97EF) or first instar larvae (betaTub56DNP0949/Df(2R)BSC782). The larvae were collected in a drop of 150 μl Schneider’s medium on a glass slide that had been pre-coated with 0.5 mg/ml concanavaline A (Sigma, C7275) in phosphate buffer (9 mM NaH2PO4, 1 mM Na2HPO4, 1 mM CaCl2, pH 6.0). Larval cuticles were gently ruptured with forceps. This allowed migration of hemocytes out of the larvae and their subsequent initial attachment to the cover slip. After 15 minutes at room temperature, larvae were discarded. Before fixation or live imaging, hemocytes were allowed to spread during a 60 minute incubation at 25°C. In case of colcemid treatment, the compound was added to final concentration 10 μM in Schneider’s medium. Hemocytes were fixed either with 4% paraformaldehyde in phosphate buffered saline (PBS) at room temperature or (for Fig. 6D) with methanol containing 1 mM EGTA for 10 minutes at −20°C (Hoogenraad et al., 2000).
Images from fixed samples were acquired with an Olympus FluoView 1000 laser-scanning confocal microscope with a 60x/1.35 objective (Figs 2C, 3D, 4C,D, 6D,E, 7A,B), with a 40x/1.3 objective (Sup. Figs. 3B,C, 5) or with a Zeiss Cell Observer HS wide-field microscope with a 63x/1.4 objective (Figs 2B,D, 6A,B), or with 16x/0.5 objective (Fig. 5C). Z-stacks were acquired. Single sections are presented in all images, except in Fig. 3A, where maximum intensity projections of relevant regions made with Imaris (Bitplane) are displayed. The intensities of anti-tubulin signals in hindgut in Fig. 4C-E were measured with ImageJ in maximum projections of 5 slices taken within the middle of the hindgut tube. The intensities of anti-alpha-tubulin staining in hindgut and other embryonic tissues (Fig. 7) were measured in single slices with ImageJ after manual selection of the regions of interest. Image z-stacks of eyes (Fig. 5B) were acquired with a Zeiss AxioCam HRc camera and converted into extended focus images using Helicon Focus Software (Helicon Soft Ltd.).
Time lapse imaging of EB1-GFP comets in live hemocytes was done with a VisiScope spinning disc confocal microscope (Visitron) on an Olympus IX83 microscope stand with a 100x/1.4 objective. Three sections with 0.1 μm spacing were acquired at 1 sec intervals. Signals in case of R-97EF and S-97EF were deconvolved using Huygens Software (Scientific Volume Imaging b.v.). Quantification of microtubule dynamics after sum of intensity projections was done with u-track software (Jaqaman et al., 2008). To differentiate the moving EB1-GFP comet signals from the stationary EB1-GFP signals characteristically present in the R-97EF and S-97EF hemocytes along microtubule bundles, image sequences were processed arithmetically in ImageJ as follows. In a first variant image sequence, the first four frames were deleted. The last four frames were deleted in a second variant. By subtracting variant 2 from variant 1, we arrived at an image sequence from which background was subtracted (Image J, rolling ball radius 2 pixels) before analysis with u-track.
Characterization of the hindgut ultrastructure in w1 and betaTub97EFMiMIC after development at 25°C to 15-16 hours AED by transmission electron microscopy and FIBSEM was achieved as described in detail in Supplementary information.
Competing interests
No competing interests declared.
Author contributions
F.M. performed all experiments (except microarray analyses and part of electron microscopy), analyzed data, designed experiments together with C.F.L. and contributed to manuscript writing. F.B. performed expression profiling. J.B. supported the generation of transgenic strains. M.K. performed electron microscopy, C.F.L conceived and supervised the study, analyzed data, and wrote the manuscript.
Funding
This work was supported by the Swiss National Science Foundation (grant 31003A_152667 to C.F.L.).
Acknowledgements
We thank Renate Renkawitz-Pohl for anti-beta-Tubulin 60D and Nikola Linsi for his contributions to antibody characterization. We are also grateful to Rita Lecca and the Functional Genomics Center Zurich for supporting microarray hybridizations, to Martin Moser for qRT-PCR support, as well as to Andres Käch and the Center for Microscopy and Image Analysis (University of Zurich).