a-burrowingtunneling-assay-for-detection-hypoxia-drosophila

A Burrowing/Tunneling Assay for Detection of Hypoxia in <em>Drosophila melanogaster</em> Larvae

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen ...

Rice University

A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM.

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila.

Drosophila melanogaster has been intensely studied for almost 100 years. The sophisticated array of genetic and molecular tools that have evolved for analysis of gene function in this organism are unique. Further, Drosophila is a complex multi-cellular organism in which many aspects of development and behavior parallel those in human beings. These combined advantages have permitted research in Drosophila to make seminal contributions to the understanding of fundamental biological processes and ensure that Drosophila will continue to provide unique insights in the genomic era. An overview of the genetic methodologies available in Drosophila is given here, together with examples of outstanding recent contributions of Drosophila to our understanding of cell and organismal biology. The growing contribution of Drosophila to our knowledge of gravity-related responses is addressed.

Drosophila melanogaster--the model organism of choice for the complex biology of multi-cellular organisms.

Gravity is a constant stimulus for life on Earth and most organisms have evolved structures to sense gravitational force and incorporate its influence into their behavioral repertoire. Here we focus attention on animals and their diverse structures for perceiving and responding to the gravitational vector-one of the few static reference stimuli for any mobile organism. We discuss vertebrate, arthropod, and nematode models from the perspective of the role that genetics is playing in our understanding of graviperception in each system. We describe the key sensory structures in each class of organism and present what is known about the genetic control of development of these structures and the molecular signaling pathways operating in the mature organs. We also discuss the role of large genetic screens in identifying specific genes with roles in mechanosensation and graviperception.

Genetics of graviperception in animals.

Androcam is a Drosophila melanogaster calmodulin-related protein that functions specifically in the testis. We show that the Acam gene is part of a cluster of three intronless genes arranged in a head-to-tail manner. The additional genes also encode calmodulin-related proteins with testis-specific transcription. Acam and the 5'-most gene (gene1) generate monocistronic transcripts. Surprisingly, the central gene (gene2) is transcribed only as a dicistronic transcript with Acam. A similar cluster is found in D. yakuba. In D. pseudoobscura, the cluster contains four genes: two Acam-type genes downstream of a single gene related to both gene1 and gene2 and a fourth weakly related gene. Nevertheless, the D. pseudoobscura cluster also generates a dicistronic transcript from a gene pair analogous to the gene2-Acam pair. A cotranscribed gene1/2-Acam gene pair may be the founding feature of this locus. Although Acam protein is present in D. melanogaster and D. pseudoobscura testes, cognate proteins for the gene1/2-type ORFs are not detectable by immunoblotting and mass spectrometry techniques.

A dicistronic gene pair within a cluster of "EF-hand" protein genes in the genomes of Drosophila species.

The ability of near-infrared fluorescence imaging to detect single-walled carbon nanotubes (SWNTs) in organisms and biological tissues has been explored using Drosophila melanogaster (fruit flies). Drosophila larvae were raised on food containing approximately 10 ppm of disaggregated SWNTs. Their viability and growth were not reduced by nanotube ingestion. Near-IR nanotube fluorescence was imaged from intact living larvae, and individual nanotubes in dissected tissue specimens were imaged, structurally identified, and counted to estimate a biodistribution.

Single-walled carbon nanotubes in the intact organism: near-IR imaging and biocompatibility studies in Drosophila.

Males of the genus Drosophila produce sperm of remarkable length. Investigation of giant sperm production in Drosophila melanogaster has demonstrated that specialized actin and microtubule structures play key roles. The gene yuri gagarin (yuri) encodes a novel protein previously identified through its role in gravitaxis. A male-sterile mutation of yuri has revealed roles for Yuri in the functions of the actin and tubulin structures of spermatogenesis. Yuri is a component of the motile actin cones that individualize the spermatids and is essential for their formation. Furthermore, Yuri is required for actin accumulation in the dense complex, a microtubule-rich structure on the sperm nuclei thought to strengthen the nuclei during elongation. In the yuri mutant, late clusters of syncytial nuclei are deformed and disorganized. The basal bodies are also mispositioned on the nuclei, and the association of a specialized structure, the centriolar adjunct (CA), with the basal body is lost. Some of these nuclear defects might underlie a further unexpected abnormality: sperm nuclei occasionally locate to the wrong ends of the spermatid cysts. The structure of the axonemes that grow out from the basal bodies is affected in the yuri mutant, suggesting a possible role for the CA in axoneme formation.

Yuri gagarin is required for actin, tubulin and basal body functions in Drosophila spermatogenesis.

Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.

The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

Controlled chemical modifications of single-walled carbon nanotubes (SWCNTs) that tune their useful properties have been sought for multiple applications. We found that beneficial optical changes in SWCNTs resulted from introducing low concentrations of oxygen atoms. Stable covalently oxygen-doped nanotubes were prepared by exposure to ozone and then light. Treated samples showed distinct, structure-specific near-infrared fluorescence at wavelengths 10 to 15% longer than displayed by pristine semiconducting SWCNTs. Dopant sites harvest light energy absorbed in undoped nanotube regions by trapping mobile excitons. The oxygen-doped SWCNTs are much easier to detect and image than pristine SWCNTs because they give stronger near-infrared emission and do not absorb at the shifted emission wavelength.

Oxygen doping modifies near-infrared band gaps in fluorescent single-walled carbon nanotubes.

The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin "cones" that mediate spermatid individualization. We used the tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuri(F64), failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions.

Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin.

Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. We show that the androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The androcam N lobe noncanonically binds a single Ca(2+) and is locked in a "closed" conformation, causing androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, androcam replacing calmodulin at IQ will abolish a Ca(2+)-regulated, calmodulin-mediated myosin VI structural change. We propose that the N lobe prevents androcam from interfering with other calmodulin-mediated Ca(2+) signaling events. We discuss how gene duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins.

Structure of androcam supports specialized interactions with myosin VI.

Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB (Bric-a-brac,Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In lov(47) , Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. lov(47) mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult lov(47) males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. lov(47) adults also show more defective negative gravitaxis than the previously isolated lov(91Y) mutant. In contrast, lov(66) produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We propose a negative regulatory role for the DNA deleted in lov(66) .

The Drosophila BTB domain protein Jim Lovell has roles in multiple larval and adult behaviors.

Space travel presents unlimited opportunities for exploration and discovery, but requires better understanding of the biological consequences of long-term exposure to spaceflight. Immune function in particular is relevant for space travel. Human immune responses are weakened in space, with increased vulnerability to opportunistic infections and immune-related conditions. In addition, microorganisms can become more virulent in space, causing further challenges to health. To understand these issues better and to contribute to design of effective countermeasures, we used the Drosophila model of innate immunity to study immune responses in both hypergravity and spaceflight. Focusing on infections mediated through the conserved Toll and Imd signaling pathways, we found that hypergravity improves resistance to Toll-mediated fungal infections except in a known gravitaxis mutant of the yuri gagarin gene. These results led to the first spaceflight project on Drosophila immunity, in which flies that developed to adulthood in microgravity were assessed for immune responses by transcription profiling on return to Earth. Spaceflight alone altered transcription, producing activation of the heat shock stress system. Space flies subsequently infected by fungus failed to activate the Toll pathway. In contrast, bacterial infection produced normal activation of the Imd pathway. We speculate on possible linkage between functional Toll signaling and the heat shock chaperone system. Our major findings are that hypergravity and spaceflight have opposing effects, and that spaceflight produces stress-related transcriptional responses and results in a specific inability to mount a Toll-mediated infection response.

Toll mediated infection response is altered by gravity and spaceflight in Drosophila.

Peptidergic neurons are a group of neuronal cells that synthesize and secrete peptides to regulate a variety of biological processes. To identify genes controlling the development and function of peptidergic neurons, we conducted a screen of 545 splice-trap lines and identified 28 loci that drove expression in peptidergic neurons when crossed to a GFP reporter transgene. Among these lines, an insertion in the alan shepard (shep) gene drove expression specifically in most peptidergic neurons. shep transcripts and SHEP proteins were detected primarily and broadly in the central nervous system (CNS) in embryos, and this expression continued into the adult stage. Loss of shep resulted in late pupal lethality, reduced adult life span, wing expansion defects, uncoordinated adult locomotor activities, rejection of males by virgin females, and reduced neuropil area and reduced levels of multiple presynaptic markers throughout the adult CNS. Examination of the bursicon neurons in shep mutant pharate adults revealed smaller somata and fewer axonal branches and boutons, and all of these cellular phenotypes were fully rescued by expression of the most abundant wild-type shep isoform. In contrast to shep mutant animals at the pharate adult stage, shep mutant larvae displayed normal bursicon neuron morphologies. Similarly, shep mutant adults were uncoordinated and weak, while shep mutant larvae displayed largely, although not entirely, normal locomotor behavior. Thus, shep played an important role in the metamorphic development of many neurons.

Neuronal remodeling during metamorphosis is regulated by the alan shepard (shep) gene in Drosophila melanogaster.

Nanomaterials with luminescence in the short-wave infrared (SWIR) region are of special interest for biological research and medical diagnostics because of favorable tissue transparency and low autofluorescence backgrounds in that region. Single-walled carbon nanotubes (SWCNTs) show well-known sharp SWIR spectral signatures and therefore have potential for noninvasive detection and imaging of cancer tumours, when linked to selective targeting agents such as antibodies. However, such applications face the challenge of sensitively detecting and localizing the source of SWIR emission from inside tissues. A new method, called spectral triangulation, is presented for three dimensional (3D) localization using sparse optical measurements made at the specimen surface. Structurally unsorted SWCNT samples emitting over a range of wavelengths are excited inside tissue phantoms by an LED matrix. The resulting SWIR emission is sampled at points on the surface by a scanning fibre optic probe leading to an InGaAs spectrometer or a spectrally filtered InGaAs avalanche photodiode detector. Because of water absorption, attenuation of the SWCNT fluorescence in tissues is strongly wavelength-dependent. We therefore gauge the SWCNT-probe distance by analysing differential changes in the measured SWCNT emission spectra. SWCNT fluorescence can be clearly detected through at least 20 mm of tissue phantom, and the 3D locations of embedded SWCNT test samples are found with sub-millimeter accuracy at depths up to 10 mm. Our method can also distinguish and locate two embedded SWCNT sources at distinct positions.

Spectral triangulation: a 3D method for locating single-walled carbon nanotubes in vivo.

The Drosophila protein Jim Lovell (Lov) is a putative transcription factor of the BTB/POZ (Bric- a-Brac/Tramtrack/Broad/ Pox virus and Zinc finger) domain class that is expressed in many elements of the developing larval nervous system. It has roles in innate behaviors such as larval locomotion and adult courtship. In performing tissue-specific knockdown with the Gal4-UAS system we identified a new behavioral phenotype for lov: larvae failed to burrow into their food during their growth phase and then failed to tunnel into an agarose substratum during their wandering phase. We determined that these phenotypes originate in a previously unrecognized role for lov in the tracheae. By using tracheal-specific Gal4 lines, Lov immunolocalization and a lov enhancer trap line, we established that lov is normally expressed in the tracheae from late in embryogenesis through larval life. Using an assay that monitors food burrowing, substrate tunneling and death we showed that lov tracheal knockdown results in tracheal fluid-filling, producing hypoxia that activates the aberrant behaviors and inhibits development. We investigated the role of lov in the tracheae that initiates this sequence of events. We discovered that when lov levels are reduced, the tracheal cells are smaller, more numerous and show lower levels of endopolyploidization. Together our findings indicate that Lov is necessary for tracheal endoreplicative growth and that its loss in this tissue causes loss of tracheal integrity resulting in chronic hypoxia and abnormal burrowing and tunneling behavior.

Article	Year
Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila. Protein science : a publication of the Protein Society\| PubMed ID: 15557269	2004
Drosophila melanogaster--the model organism of choice for the complex biology of multi-cellular organisms. Gravitational and space biology bulletin : publication of the American Society for Gravitational and Space Biology\| PubMed ID: 16038090	2005
Genetics of graviperception in animals. Advances in genetics\| PubMed ID: 16291213	2005
A dicistronic gene pair within a cluster of "EF-hand" protein genes in the genomes of Drosophila species. Genomics\| PubMed ID: 16750900	2006
Single-walled carbon nanotubes in the intact organism: near-IR imaging and biocompatibility studies in Drosophila. Nano letters\| PubMed ID: 17696559	2007
Yuri gagarin is required for actin, tubulin and basal body functions in Drosophila spermatogenesis. Journal of cell science\| PubMed ID: 18477609	2008
The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus. Journal of cell science\| PubMed ID: 20647369	2010
Oxygen doping modifies near-infrared band gaps in fluorescent single-walled carbon nanotubes. Science (New York, N.Y.)\| PubMed ID: 21109631	2010
Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin. Experimental cell research\| PubMed ID: 21126519	2011
Structure of androcam supports specialized interactions with myosin VI. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22851764	2012
The Drosophila BTB domain protein Jim Lovell has roles in multiple larval and adult behaviors. PloS one\| PubMed ID: 23620738	2013
Toll mediated infection response is altered by gravity and spaceflight in Drosophila. PloS one\| PubMed ID: 24475130	2014
Neuronal remodeling during metamorphosis is regulated by the alan shepard (shep) gene in Drosophila melanogaster. Genetics\| PubMed ID: 24931409	2014
Spectral triangulation: a 3D method for locating single-walled carbon nanotubes in vivo. Nanoscale\| PubMed ID: 27140495	2016
Failure to Burrow and Tunnel Reveals Roles for jim lovell in the Growth and Endoreplication of the Drosophila Larval Tracheae. PloS one\| PubMed ID: 27494251	2016

Kathleen M. Beckingham

Biography

JoVE Journal Publications

Other Publications