Systematic characterization of Rab GTPase cell type expression and subcellular localization in Drosophila melanogaster

The Rab family of small GTPases orchestrates intracellular endomembrane transport through the recruitment of diverse effector proteins. Since its first discovery in 1987, almost 70 Rab proteins have been identified in humans to date and their perturbed function is implicated in several hereditary and acquired diseases. In this Ph.D. thesis, I systematically characterize cell type expression and subcellular localization of all Rab proteins present in Drosophila melanogaster utilizing a genetic resource that represents a major advance for studying membrane trafficking in vivo: the ’Drosophila YRab library’. This collection comprises 27 different D. melanogaster knock-in lines that harbor YFPMyc fusions to each Rab protein, referred to as YRab. For each YRab, I present a comprehensive data set of quantitative and qualitative expression profiles across six larval and adult tissues that include 23 annotated cell types. The whole image data set, along with its annotations, is publicly accessible through the FLYtRAB database that links to CATMAID for online browsing of tissues. I exploit this data set to address basic cell biological questions. i) How do differentiating cells reorganize their transport machinery to perform cell type-specific functions? My data indicates that qualitative and quantitative changes in YRab protein expression facilitate the functional specialization of differentiated cells. I show that about half of the YRab complement is ubiquitously expressed across D. melanogaster tissues, while others are missing from some cell types or reflect strongly restricted cell type expression, e.g. in the nervous system. I also depict that relative YRab expression levels change as cells differentiate. ii) Are specific Rab proteins dedicated to apical or basolateral protein transport in all epithelia? My data suggests that the endomembrane architecture reflects specific tasks performed by particular epithelial tissues, rather than a generalized apicobasal organization. I demonstrate that there is no single YRab that is similarly polarized in all epithelia. Rather, different epithelial tissues dynamically polarize the subcellular localization of many YRab compartments, producing membrane trafficking architectures that are tissue- and stage-specific. I further discuss YRab cell type expression and subcellular localization in the context of Rab family evolution. I report that the conservation of YRab protein expression across D. melanogaster cell types reflects their evolutionary conservation in eukaryotes. In addition, my data supports the assumption that the flexible deployment of an expanded Rab family triggered cell differentiation in metazoans. The FLYtRAB database and the ’Drosophila Rab Library’ are complementary resources that facilitate functional predictions based on YRab cell type expression and subcellular localization, and to subsequently test them by genetic loss-of-function experiments. I demonstrate the power of this approach by revealing new and redundant functions for Rab23 and Rab35 in wing vein patterning. My data collectively highlight that in vivo studies of endomembrane transport pathways in different D. melanogaster cell types is a valuable approach to elucidate functions of Rab family proteins and their potential implications for human disease

Adipose tissue ATGL modifies the cardiac lipidome in pressure-overload-induced left ventricular failure

Adipose tissue lipolysis occurs during the development of heart failure as a consequence of chronic adrenergic stimulation. However, the impact of enhanced adipose triacylglycerol hydrolysis mediated by adipose triglyceride lipase (ATGL) on cardiac function is unclear. To investigate the role of adipose tissue lipolysis during heart failure, we generated mice with tissue-specific deletion of ATGL (atATGL-KO). atATGL-KO mice were subjected to transverse aortic constriction (TAC) to induce pressure-mediated cardiac failure. The cardiac mouse lipidome and the human plasma lipidome from healthy controls (n = 10) and patients with systolic heart failure (HFrEF, n = 13) were analyzed by MS-based shotgun lipidomics. TAC-induced increases in left ventricular mass (LVM) and diastolic LV inner diameter were significantly attenuated in atATGL-KO mice compared to wild type (wt) -mice. More importantly, atATGL-KO mice were protected against TAC-induced systolic LV failure. Perturbation of lipolysis in the adipose tissue of atATGL-KO mice resulted in the prevention of the major cardiac lipidome changes observed after TAC in wt-mice. Profound changes occurred in the lipid class of phosphatidylethanolamines (PE) in which multiple PE-species were markedly induced in failing wt-hearts, which was attenuated in atATGL-KO hearts. Moreover, selected heart failure-induced PE species in mouse hearts were also induced in plasma samples from patients with chronic heart failure. TAC-induced cardiac PE induction resulted in decreased PC/PE-species ratios associated with increased apoptotic marker expression in failing wt-hearts, a process absent in atATGL-KO hearts. Perturbation of adipose tissue lipolysis by ATGL-deficiency ameliorated pressure-induced heart failure and the potentially deleterious cardiac lipidome changes that accompany this pathological process, namely the induction of specific PE species. Non-cardiac ATGL-mediated modulation of the cardiac lipidome may play an important role in the pathogenesis of chronic heart failure

Systematic characterization of Rab GTPase cell type expression and subcellular localization in Drosophila melanogaster

The Rab family of small GTPases orchestrates intracellular endomembrane transport through the recruitment of diverse effector proteins. Since its first discovery in 1987, almost 70 Rab proteins have been identified in humans to date and their perturbed function is implicated in several hereditary and acquired diseases. In this Ph.D. thesis, I systematically characterize cell type expression and subcellular localization of all Rab proteins present in Drosophila melanogaster utilizing a genetic resource that represents a major advance for studying membrane trafficking in vivo: the ’Drosophila YRab library’. This collection comprises 27 different D. melanogaster knock-in lines that harbor YFPMyc fusions to each Rab protein, referred to as YRab. For each YRab, I present a comprehensive data set of quantitative and qualitative expression profiles across six larval and adult tissues that include 23 annotated cell types. The whole image data set, along with its annotations, is publicly accessible through the FLYtRAB database that links to CATMAID for online browsing of tissues. I exploit this data set to address basic cell biological questions. i) How do differentiating cells reorganize their transport machinery to perform cell type-specific functions? My data indicates that qualitative and quantitative changes in YRab protein expression facilitate the functional specialization of differentiated cells. I show that about half of the YRab complement is ubiquitously expressed across D. melanogaster tissues, while others are missing from some cell types or reflect strongly restricted cell type expression, e.g. in the nervous system. I also depict that relative YRab expression levels change as cells differentiate. ii) Are specific Rab proteins dedicated to apical or basolateral protein transport in all epithelia? My data suggests that the endomembrane architecture reflects specific tasks performed by particular epithelial tissues, rather than a generalized apicobasal organization. I demonstrate that there is no single YRab that is similarly polarized in all epithelia. Rather, different epithelial tissues dynamically polarize the subcellular localization of many YRab compartments, producing membrane trafficking architectures that are tissue- and stage-specific. I further discuss YRab cell type expression and subcellular localization in the context of Rab family evolution. I report that the conservation of YRab protein expression across D. melanogaster cell types reflects their evolutionary conservation in eukaryotes. In addition, my data supports the assumption that the flexible deployment of an expanded Rab family triggered cell differentiation in metazoans. The FLYtRAB database and the ’Drosophila Rab Library’ are complementary resources that facilitate functional predictions based on YRab cell type expression and subcellular localization, and to subsequently test them by genetic loss-of-function experiments. I demonstrate the power of this approach by revealing new and redundant functions for Rab23 and Rab35 in wing vein patterning. My data collectively highlight that in vivo studies of endomembrane transport pathways in different D. melanogaster cell types is a valuable approach to elucidate functions of Rab family proteins and their potential implications for human disease

Systematic characterization of Rab GTPase cell type expression and subcellular localization in Drosophila melanogaster

The Rab family of small GTPases orchestrates intracellular endomembrane transport through the recruitment of diverse effector proteins. Since its first discovery in 1987, almost 70 Rab proteins have been identified in humans to date and their perturbed function is implicated in several hereditary and acquired diseases. In this Ph.D. thesis, I systematically characterize cell type expression and subcellular localization of all Rab proteins present in Drosophila melanogaster utilizing a genetic resource that represents a major advance for studying membrane trafficking in vivo: the ’Drosophila YRab library’. This collection comprises 27 different D. melanogaster knock-in lines that harbor YFPMyc fusions to each Rab protein, referred to as YRab. For each YRab, I present a comprehensive data set of quantitative and qualitative expression profiles across six larval and adult tissues that include 23 annotated cell types. The whole image data set, along with its annotations, is publicly accessible through the FLYtRAB database that links to CATMAID for online browsing of tissues. I exploit this data set to address basic cell biological questions. i) How do differentiating cells reorganize their transport machinery to perform cell type-specific functions? My data indicates that qualitative and quantitative changes in YRab protein expression facilitate the functional specialization of differentiated cells. I show that about half of the YRab complement is ubiquitously expressed across D. melanogaster tissues, while others are missing from some cell types or reflect strongly restricted cell type expression, e.g. in the nervous system. I also depict that relative YRab expression levels change as cells differentiate. ii) Are specific Rab proteins dedicated to apical or basolateral protein transport in all epithelia? My data suggests that the endomembrane architecture reflects specific tasks performed by particular epithelial tissues, rather than a generalized apicobasal organization. I demonstrate that there is no single YRab that is similarly polarized in all epithelia. Rather, different epithelial tissues dynamically polarize the subcellular localization of many YRab compartments, producing membrane trafficking architectures that are tissue- and stage-specific. I further discuss YRab cell type expression and subcellular localization in the context of Rab family evolution. I report that the conservation of YRab protein expression across D. melanogaster cell types reflects their evolutionary conservation in eukaryotes. In addition, my data supports the assumption that the flexible deployment of an expanded Rab family triggered cell differentiation in metazoans. The FLYtRAB database and the ’Drosophila Rab Library’ are complementary resources that facilitate functional predictions based on YRab cell type expression and subcellular localization, and to subsequently test them by genetic loss-of-function experiments. I demonstrate the power of this approach by revealing new and redundant functions for Rab23 and Rab35 in wing vein patterning. My data collectively highlight that in vivo studies of endomembrane transport pathways in different D. melanogaster cell types is a valuable approach to elucidate functions of Rab family proteins and their potential implications for human disease

Validation of the phenotype of <i>Eph</i> RNAi lines.

<p><b>A.</b> Exon-intron structure of the <i>Eph</i> gene and regions targeted by the indicated RNAi lines. <b>B–D.</b> Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting <i>Eph</i> using the RNAi lines (B) 4771, (C) GL01189 or (D) GL00192. The clones of cells are identified by co-expression of DsRed (red); cells of the posterior compartment are labeled by expression of Venus under control of the <i>engrailed</i> gene (<i>en-</i>Venu<i>s</i>, green). Two clones expressing <i>Eph^dsRNA</i> located in different compartments sharing a common interface along the AP boundary locally distort the shape of the AP boundary (asterisks). In (B), <i>dcr2</i> is co-expressed to increase the efficiency of RNAi. <b>E.</b> Number of cases where two clones expressing double-stranded RNA targeting <i>Eph</i> located in different compartments sharing a common interface along the AP boundary distort the AP boundary towards the posterior side (A>P), towards the anterior side (P>A), or do not distort the AP boundary (no effect). Scale bar is 50 µm.</p

Summary of RNA interference screen.

<p><b>A.</b> Number of RNAi lines screened categorized for the class of targeted gene products. <b>B.</b> Number of the analyzed RNAi lines and fractions of the phenotypic categories as a percentage.</p

Clones of cells expressing double-stranded RNA targeting the <i>Eph</i> gene distort the AP boundary.

<p><b>A–C.</b> Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting (A–B) <i>Eph</i> or (C) <i>FasIII.</i> The clones of cells are identified by co-expression of DsRed (red); cells of the posterior compartment are labeled by expression of Venus under control of the <i>engrailed</i> gene (<i>en-</i>Venus, green). (A) Individual clones abutting the AP boundary from either the A or P compartment do not influence the shape of the boundary (asterisk). (B) Two clones expressing <i>Eph^dsRNA</i> (ID-Number 4771 (VDRC)) located in different compartments sharing a common interface along the AP boundary locally distort the shape of the AP boundary (asterisks). (C) Two clones expressing <i>FasIII^dsRNA</i> located in different compartments sharing a common interface along the AP boundary do not distort the shape of the AP boundary (asterisks). Scale bar is 50 µm.</p

An RNA Interference Screen for Genes Required to Shape the Anteroposterior Compartment Boundary in <i>Drosophila</i> Identifies the Eph Receptor

<div><p>The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in <i>Drosophila</i> wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in <i>Drosophila</i> and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.</p></div

An assay to identify genes required to maintain the straight shape of the AP boundary.

<p>A. Scheme of tester stock and cross to generate clones of cell expressing double-stranded RNA. The tester stock contains a <i>Flp</i> recombinase transgene under control of the heat-shock inducible promoter hsp70 (<i>hs</i>) on chromosome I <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114340#pone.0114340-Struhl1" target="_blank">[36]</a> and the <i>engrailed-Venus</i> transgene on chromosome II (not shown). Chromosome III contains a transgene composed of the <i>Act5C</i> promoter followed by a FRT site, the coding sequence for the mouse transmembrane protein CD2 followed by a transcriptional terminator (Term), a second FRT site, and finally Gal4. We have recombined on the same chromosome a transgene expressing DsRed under control of UAS sequences (not shown). The tester stock is crossed to individual RNAi fly lines. The resulting progeny larvae are heat-shocked to express the Flp recombinase that in turn induces recombination between the two FRT sites on the tester stock. This recombination places Gal4 under the direct control of the <i>Act5C</i> promoter leading to Gal4 expression specifically in the clones of cells. Gal4 then binds to the UAS sequences in the RNAi line and directs expression of double-stranded RNA that leads to RNA interference. At the same time, Gal4 will induce expression of DsRed (not shown) in the same cells. B. Scheme of the assay. The central part of the wing imaginal disc is schematically drawn as an oval. A Venus fluorescent protein (green) is specifically expressed in all cells of the posterior compartment under control of the promoter sequences of the <i>engrailed</i> gene (<i>en</i>-Venus). Clones of cells expressing double stranded RNA are identified by co-expression of DsRed (red). Examples on the left depict wild-type clones located in the anterior (A) or posterior (P) compartments. Examples on the right depict a anterior clone mis-segregating into the posterior territory (top) and an posterior clone mis-segregating into the anterior territory (bottom) of the wing disc. Clones co-expressing DsRed and Venus are depicted in yellow.</p

Power-duration relationship comparison in competition sprint cyclists from 1-s to 20-min. Sprint performance is more than just peak power.

Current convention place peak power as the main determinant of sprint cycling performance. This study challenges that notion and compares two common durations of sprint cycling performance with not only peak power, but power out to 20-min. There is also a belief where maximal efforts of longer durations will be detrimental to sprint cycling performance. 56 data sets from 27 cyclists (21 male, 6 female) provided maximal power for durations from 1-s to 20-min. Peak power values are compared to assess the strength of correlation (R2), and any relationship (slope) across every level. R2 between 15-s- 30-s power and durations from 1-s to 20-min remained high (R2 ≥ 0.83). Despite current assumptions around 1-s power, our data shows this relationship is stronger around competition durations, and 1-s power also still shared strong relationships with longer durations out to 20-min. Slopes for relationships at shorter durations were closer to a 1:1 relationship than longer durations, but closer to long-duration slopes than to a 1:1 line. The present analyses contradicts both well-accepted hypotheses that peak power is the main driver of sprint cycling performance and that maximal efforts of longer durations out to 20-min will hinder sprint cycling. This study shows the importance and potential of training durations from 1-s to 20-min over a preparation period to improve competition sprint cycling performance