Ion pumps in Drosophila hearing

Transcription

1 University of Iowa Iowa Research Online Theses and Dissertations 2015 Ion pumps in Drosophila hearing Betul Zora University of Iowa Copyright 2015 Betul Zora This dissertation is available at Iowa Research Online: Recommended Citation Zora, Betul. "Ion pumps in Drosophila hearing." MS (Master of Science) thesis, University of Iowa, Follow this and additional works at: Part of the Biology Commons

2 ION PUMPS IN DROSOPHILA HEARING by Betul Zora A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Biology in the Graduate College of The University of Iowa August 2015 Thesis Supervisor: Professor Daniel F. Eberl

3 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL This is to certify that the Master s thesis of MASTER S THESIS Betul Zora has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Biology at the August 2015 graduation. Thesis Committee: Daniel F. Eberl. Thesis Supervisor John Manak Alan Kay

4 To Ayhan and Yildiz Zora, my father and my mother for supporting me through the hardest challenge I have ever undertaken ii

5 ACKNOWLEDGEMENTS This work would not have been possible without the patience and guidance of Dr. Eberl, who could help me regain my focus when I felt overwhelmed by the scope of my work. And I want to thank him for providing me with the resources and the environment I needed to complete my work. With his help, I am a better scientist. I want to also thank other members of my thesis committee, Dr. Manak and Dr. Kay for sharing their feedback and insight. I want to thank current and past members of the Eberl lab for all of their help and encouragement. Specifically, I want to thank Elena Sivan-Loukianova for her advice on all matters scientific and otherwise. Her experience in microscopy was an invaluable resource. I want to thank Julie Jacobs and Kevin Christie for sharing their experience in electrophysiology with me. I want to thank Kevin for the microarray data that was a key filter for the present work. I want to thank Madhuparna Roy for her work on the Na/K pump, upon which I built my own project. And the numerous undergraduate students who frequently listened to my grievances. I want to thank Andrew Zelhoff for sharing his EYS-GFP fusion protein, which provided a comparison for the localization of some of the proteins I studied. I want to extend a special thanks to Julian Dow for sharing reagents and the other fluorescently tagged fly lines reported here. I want to thank the Bloomington Drosophila Stock Center and the Vienna Drosophila Resource Center for the numerous RNAi lines that was so central to this project. I want to thank the Iowa Center for Molecular Auditory Neuroscience for providing the resources and facility I needed to complete my work. Finally, I want to thank my family as well as friends in the graduate college. iii

6 ABSTRACT Ion pumps establish homeostasis across the membranes of living cells. Hearing is a mechanotransduction event that takes place in a closed compartment containing a fluid high in K + concentrations. In Drosophila melanogaster, this closed compartment is formed by a scolopale cell that wraps around the dendrite of sensory neurons. The receptor lymph is maintained by the scolopale cell. The lumenal membrane of the scolopale cell is the wall of the compartment containing the receptor lymph, the scolopale space. The ablumenal membrane of the scolopale cell creates the border of the scolopidium. The Na/K pump is located on the ablumenal membrane of the scolopale cell, bringing K + into the scolopale cell cytoplasm and extruding K electrogenically (Roy et al, 2013). We explored other primary and secondary ion pumps that are involved in creating a K + -rich lumen in the Malpighian tubule (Day et al, 2008; Rodan et al, 2012). We used RNAi technology to knockdown one gene at a time and electrophysiology to measure a sound evoked potential (SEP) that reflects the fly s ability to hear. We found that knocking down V-ATPase, a proton pump, subunits involved in proton extrusion significantly reduces the SEP of knockdown flies. The involvement of cation chloride cotransporters (CCCs) and cation proton antiporter (CPAs), both secondary ion pumps that use the gradients created by the Na/K pump and V-ATPase respectively to pump other ions up their gradient, is less clear. We found that knocking down Nhe3, a CPA, significantly reduced the SEP when knocked down in the scolopale cell, suggesting it as a partner to the V-ATPase. Knocking down CG31547, a CCC, statistically increased the SEP, possibly a type1 statistical error. iv

7 PUBLIC ABSTRACT The organ the fruitfly uses to hear contains a fluid called the receptor lymph. Cells use ion pumps to maintain ion concentrations in fluid-filled compartments. Here, cells surrounding the receptor lymph were genetically manipulated so that they do not produce each of three kinds of ion pumps. This will let us test if they are important in fly hearing. These pumps are also important for human hearing so understanding how these pumps work may help understand and perhaps prevent hearing loss. We explored the proton pump, which acidifies specialized compartments, and found it to be important in mediating hearing. The second type of pump, the cation proton antiporters (CPAs) use the energy provided by the proton pump to produce a compartment concentrated in an ion key for fruitfly (and human) hearing. And the third kind of pump was called the cation chloride cotransporters (CCCs). We found that flies lacking Nhe3, a kind of CPA, in the structures responsible for making the receptor lymph were not able to hear as well. In addition, Nha2, another type of CPA, is located in the specialized cells maintaining the receptor lymph; this candidate we could not test functionally. In conclusion, we provided evidence that the proton pump may work with Nhe3 and Nha2 to maintain proper ion concentrations in the receptor lymph, but more work needs to be done to understand how these three pumps interact. v

8 TABLE OF CONTENTS LIST OF TABLES... viii LIST OF FIGURES... ix Chapter I. Introduction... 1 A. Hearing in Drosophila melanogaster... 2 B. Structure of the Drosophila scolopidium... 3 C. NompC is the mechanosensitive ion channel in Drosophila hearing... 5 D. Ion Pumps in the Johnston s Organ... 6 Chapter II. Methods A. RNAi Knockdown RNAi constructs for the V-ATPase RNAi constructs for cation proton antiporters RNAi constructs for cation chloride cotransporters Driver lines B. Electrophysiology Rearing flies for electrophysiology Electrophysiology C. Immunohistology Solutions Fixation and cryosectioning Processing Antibodies Special flies Chapter III. Vacuolar-ATPase A. Domains of the V-ATPase complex B. V-ATPase subunits and genes expressed in the Johnston s Organ C. Changes in SEP of flies with subunits knocked down with RNAi D. Locating the V-ATPase in the Johnston s Organ Chapter IV. Secondary Transport A. Cation proton antiporters (CPAs) B. Cation chloride cotransporters (CCCs) vi

9 Chapter V. Discussion References vii

10 LIST OF TABLES Table 1 Stock center and stock number of RNAi construct flies used to target V- ATPase subunits Table 2 Stock center and stock number of RNAi flies targeting CPA's Table 3 Stock center and stock number of RNAi flies targeting CCC's Table 4 All Drosophila genes encoding V-ATPase genes, their expression in the Johnston s Organ and the significance between knockdown flies and construct controls viii

11 LIST OF FIGURES Figure 1 The Drosophila antenna Figure 2 The structure of the scolopidium and the three compartments formed by the two membranes of the scolopale cell Figure 3 The different components of the V-ATPase Figure 4 Genes encoding V-ATPase subunits expressed in the Johnston's Organt Figure 5 Subunits coded by vhaac45, vha68-2, vha36-1, vha26, vha14-1 and vha16-1 show significantly reduced SEPs when knocked down with ato-gal Figure 6 Scolopale cell specific knockdown of V-ATPase subunit genes produces significant reductions in SEP with vhaac45, vha36-1, vha26, vha100-1 and vhaac Figure 7 Canton-S antenna stained with anti vha55 (green) and TexasRed Phalloidin (red) Figure 8 Cross section of Canton-S stained with anti-vha55 gene product (green) and TexasRed Phalloidin (red) Figure 9 EYS-GFP fusion with nompa-gal4 driver (green), 22C10 (blue), TexasRed Phalloidin (red) Figure 10 Nhe3 knockdown with nompa-gal4 produces significantly reduced SEPs Figure 11 Nha2-YFP fusion (green) expressed with ato-gal Figure 12 Nha2-YFP fusion (green) expressed with nompa-gal Figure 13 The CCC family tree Figure 14 Knocking down CCC genes does not change the SEP Figure 15 The present model of ion transport in the scolopidium ix

12 ION PUMPS IN DROSOPHILA HEARING x

13 Chapter I. Introduction Ion homeostasis is an integral part of cell survival. Ion gradients are maintained across membranes with various pumps and ion transporters. There are two kinds of ion transport: primary and secondary. Primary transport mechanisms involve the pumping of ions across a membrane directly coupled to the energy of ATP hydrolysis. In this case, the Na/K pump uses ATP hydrolysis to pump Na + out of the cell and K + into the cell at a 3:2 ratio. Another example of this is the V- ATPase. The V-ATPase couples ATP hydrolysis to proton extrusion. Secondary transport mechanisms use already present ion gradients to pump other ions across the membrane. For example, the cation chloride cotransporters use the Na + and K + gradient created by the Na/K pump to transport Cl - in or out of the cell. Similarly, the cation proton antiporters using the proton gradient created by the V-ATPase to pump Na + and K + out of the cytoplasm. Both forms of transport are important for preserving the gradients across the membrane. Such ionic gradients are used in excitable cells, such as peripheral nervous system receptor neurons, in gathering and relaying information. Mechanoreception is a process that converts a mechanical signal into the electrochemical language of neurons. Hearing is a mechanosensory process that plays important biological roles in both vertebrates and invertebrates, in courtship, aggression, alarm calling and predator evasion to name a few. Across this taxonomic breadth, hearing has interesting mechanistic conservation. Presently, we explore the importance of ion balance in fruitfly hearing and this has implications on fundamental conserved mechanosensory mechanism. 1

14 A. Hearing in Drosophila melanogaster In Drosophila hearing, as in any animal, a sound wave is received externally and is converted into the movement of structures. This movement is relayed to internal mechanoreceptors that open mechanosensitive ion channels. The receptor potential formed by the opening of these channels activates a neuron and then the information is channeled into an auditory nerve, which sends it to the brain for processing. A. B. Figure 1 The Drosophila antenna. The antenna serves as the fly ear. There are four major sections to the antenna. A) a micrograph of the adult antenna. The first antennal segment (a1) connects the antenna to the head. The second antennal segment (a2) houses the Johnston s Organ, a collection of chordotonal mechanotranducers that serves as the fly auditory organ. The third antennal segment (a3) contains the fly olfactory receptors. More importantly, a3 is attached to the arista (ar), a structure that detects external sound waves and serves as the fly tympanic membrane. The movement of the ar twists a3 against a2, much like the movement of the tympanic membrane moving the three bones of the middle ear. These structures are shown on the figure on the left (adopted from Boekhoff-Falk, 2005). B) The drawing shows the relationship of the mechanosensors in a2 to the joint between a2 and a3 (adopted from Boekhoff-Falk and Eberl, 2014, fig.1) In mammalian hearing, the external receptor is the tympanic membrane. The vibration of the tympanic membrane in response to a sound wave moves the three bones of the middle ear 2

15 and eventually the basal membrane in the cochlea. The mechanoreceptors are the outer hair cells in the cochlea, who connect to spiral ganglion neurons via ribbon synapses. The external receptor in Drosophila is the feather-like arista (Figure 1). The movement of the arista in response to a sound wave moves the entire third segment (Figure 1) of the fly antenna. The twisting of the third segment stretches mechanoreceptors in the second segment (a2 in the illustration), which houses the auditory organ of Drosophila, the Johnston s Organ. The Johnston s Organ is a collection of about two hundred individual scolopidia, the mechanosensory unit (Boekhoff-Falk & Eberl, 2014). Mechanoreception requires the fluid bathing the mechanoreceptors to have special properties. The fluid bathing the mechanoreceptors in the cochlea is called the endolymph. In Drosophila, it is called the receptor lymph. They are thought to be comparable in terms of the relative concentrations of ions involved in excitability, like K + and Na + (Kernan, 2007). B. Structure of the Drosophila scolopidium The fly hearing community has focused on defining the structure and development of the the Johnston s Organ since the turn of the twenty-first century (reviewed by Boekhoff-Falk & Eberl, 2014; Caldwell & Eberl, 2002; Eberl & Boekhoff-Falk, 2007). Each scolopidium is composed of a cap structure tied to the cuticle at the articulating joint of the antenna, relaying the mechanical signal. The ciliated dendrites of the two or three neurons are tightly coupled to the cap structure. All of this is enveloped by a scolopale cell, as illustrated in Figure 1. It is thought that the stretching of the scolopidium opens the mechanosensitive TRP channel, NompC, on the dendrite (Lee et al, 2010; Sun et al, 2009). One aspect of the scolopidium that distinguishes it from the cochlea is that the generation of an action potential in the neurons of the mammalian system relies on successful synaptic 3

16 transmission between the mechanosensitive hair cells and the spiral ganglion neurons. So anything that inhibits the synapse is capable of causing sensorineural hearing loss. However, in the scolopidium, there is no synapse between the receptor and the neuron. The dendrite of the neuron is the mechanosensitive receptor. Any aberration that does not directly influence mechanotransduction and the generation of an action potential will not prevent the neuron from firing in response to a sound wave. In Drosophila auditory electrophysiology, we apply a tungsten electrode directly to the antennal nerve, which contains the axons of all the scolopidial neurons, effectively measuring the compound action potential of the neurons (Eberl & Kernan, 2011). We use the amplitude of this response to gauge the fly s ability to hear. Therefore, any defects in synaptic transmission would be downstream of the recording site and thus would not impact the auditory electrophysiology. A very important cell, called the scolopale cell, wraps around the ciliated dendrite of the receptor neurons and forms a sealed extracellular compartment using septate junctions with the cap cell and with the base of the dendrite of the neuron (Figure 1). The sealed compartment is called the scolopale space and contains the fluid, the receptor lymph, in which the mechanotransduction event takes place. The scolopale cell maintains the receptor lymph. It is thought to be high in K + and low in Na +, like the endolymph in the cochlea (Kernan, 2007). However, this is yet to be proven. Evidence for K + rich receptor lymph comes from mechanosensors in the large fly, Calliphora. High K + concentrations were observed in the campaniform organs in the haltere, a structure packed with mechanosensors with a similarly sealed compartment containing receptor lymph (Grünert & Gnatzy, 1987). 4

17 C. NompC is the mechanosensitive ion channel in Drosophila hearing NompC is a TRPN channel located on the distal tip of the dendrite (Lee et al, 2010). NompC works together with the more proximally located TRPV channels inactive (iav) and nanchung (nan) to produce the receptor potential (Kernan, 2007). iav and nan are expressed in all scolopidia of the Johnston s Organ. Although nompc-gal4 is expressed specifically to a subset of scolopidia that function in hearing (Kamikouchi et al, 2009; Yan et al, 2013; Sun et al, 2009), antibody staining for NompC stains in all scholopidia (Lee et al, 2010). Transfection studies show that NompC is permeable to K +, Na + as well as Ca 2+ ions (Yan et al, 2013). The fact that NompC is permeable to K + and is located on the distal tip on auditory scolopidia lends support to the idea that the scolopale space is K + rich and that the receptor potential is K + dependent. However, this does not discount the possibility that transduction may depend on other important cations. Though if the latter were the case, the strong K + gradient could electrically play a role in strengthening the drive of other cations through NompC. Interestingly, Gong et al (2013) found that transduction in the external sensory organ, a different mechanosensory system in the fly bristles that has interesting parallels with the scolopidia in the Johnston s Organ, that removing Cl - from the external solution abolished current in the neuron. What this implies is that NompC s response on the neuron is Cl - dependent. This is interesting because the receptor lymph is completely sealed by support cells (Kernan, 2007; Gong et al, 2013). The manipulations on the recording solution affect the supporting cell more than the neuron. The loss of current when the recording solution is depleted of Cl - ions means that the support cell is maintaining the receptor lymph in a Cl - dependent manner. 5

18 This is perfectly aligned with the postulations of the present study. Here, we explore the role of a Na + dependent cation chloride cotransporter (CCC) as a possible partner to the Na/K pump on the ablumenal membrane of the scolopale cell. If the same mechanism is conserved in the Johnston s Organ, the receptor lymph maintenance also could be Cl - dependent. Work in the Malpighian tubule shows that inhibition of the Na + dependent CCC (Ncc69) reduces K + excretion (Rodan et al, 2012). These two facts together support the idea that the receptor lymph maintenance is Cl - dependent, that the receptor lymph is K + rich and Na + dependent CCCs play a role in achieving this. D. Ion Pumps in the Johnston s Organ Most work on the scolopidium has focused on development of the general structure and the ciliated nature of the dendrite (Caldwell & Eberl, 2002; Ebacher, et al, 2007; Jarman & Groves, 2013). More recent work on traumatized flies has developed flies as a model of hearing loss, validating the fruitfly as a meaningful model for hearing (Christie & Eberl, 2014; Christie et al, 2013). Only recently has work been done on the Johnston s Organ to understand the actual physiology of the structures and ion homeostasis (Roy et al, 2013). The mentioned work focused on the Na/K pump and demonstrated that specific subunits of the Na/K pump are localized in specific cell types of the scolopidium. The loss of this ion pump via the RNAi knockdown strategy, rather than leading to the complete destruction of the tissue, shows marked abnormalities, such as the invasion of the scolopale space by mitochondria. Whether the invading mitochondria originate in the neurons or in the scolopale cell has yet to be elucidated and is not within the scope of this work. Importantly, it is known that the Na/K pump is located asymmetrically on the membrane of the scolopale cell. Specifically it is in the ablumenal membrane, forming the boundary 6

19 between the scolopale cell cytoplasm and the hemolymph. This is demonstrated in cartoon format in Figure 2. The general function of the Na/K pump is to exchange cytoplasmic Na + for extracellular K + at a three to two ratio powered by ATP hydrolysis. The fact that it is located on that particular membrane of the scolopale cell suggests that it introduces K + into the scolopidium. Figure 2 The structure of the scolopidium and the three compartments formed by the two membranes of the scolopale cell. The cartoon on the right shows the basic structures of the scolopidium (modified from Roy et al, 2013). Each scolopidium consists of two or three neurons connected to an extracellular structure called the dendritic cap via ciliated dendrites. The movement of the cap cell and the dendritic cap with the stretching of the epithelium opens mechanosensitive ion channels on the distal tip of the ciliated dendrite. The ciliated dendrite is enveloped by a support cell called the scolopale cell, which maintains the receptor lymph (colored in yellow) bathing the dendrites. Because of the way the scolopale cell wraps around the dendrites, three fluid filled compartments are formed as shown in the schematic on the left. To achieve a K + rich receptor lymph, the scolopale cell must first take K + ions into its own cytoplasm. To do this, the Na/K pump is used on the ablumenal membrane. Then the K + ion must cross the lumenal membrane to reach the receptor lymph. 7

20 Because the receptor lymph is sealed by the opposite membrane of the scolopale cell, the lumenal membrane, this suggests that the putative high K + and low Na + concentration of the receptor lymph is maintained by other ion transport mechanisms. For participating pumps on the lumenal membrane, we turn to the Malpighian tubules, an excretory and osmoregulatory system that is also sensitive to ion balance. Principal cells form the Malpighian tubule with a lumen that contains high levels of K +. The Na/K pump is located on the basal membrane of the principal cells (Wieczorek et al, 2009). The lumenal membrane of these cells has the Vacuolar-ATPase (V-ATPase), a proton pump (Day et al, 2008). The V- ATPase translocates a proton from the cytoplasm across a membrane. In some cells, the V- ATPase is localized to a vesicle inside a cell, while in others, such as the principal cells, it can be in the plasma membrane (Wieczorek et al, 1999). In this way, the V-ATPase generates a proton gradient across the lumenal membrane of the principal cells. Secondary transport mechanisms take advantage of this proton gradient to pump other ions up their concentration gradient. In the principal cell, the proton gradient is thought to be used to pump K + ions, thereby creating a K + - rich lumen. Biological systems frequently deploy similar machinery in multiple contexts. Thus, it is conceivable that a similar scenario might exist in the Johnston s Organ. The Na/K pump is located on the ablumenal membrane of the scolopale cell and is responsible for bringing K + into the scolopale cell (Roy, Sivan-Loukianova, & Eberl, 2013). It is possible that the V-ATPase may be located on the lumenal membrane and would be responsible for setting up a proton gradient that a secondary transport system could exploit to pump K + into the putatively K + -rich receptor lymph. The goal of the current work is to test the V-ATPase s impact on hearing. 8

21 Secondary transport systems are often partners to the V-ATPase and the Na/K pump. Cation proton antiporters (CPAs) take advantage of the proton gradient formed by the V-ATPase to pump other ions against their concentration gradients (Day et al, 2008; Xiang et al, 2012). There are five known Drosophila CPA genes. In the Malpighian tubule, the newly discovered subfamily of CPAs consisting of the products of two of the five genes colocalize on the apical membrane with the V-ATPase. They are thought to use the proton gradient to pump K + up its concentration gradient. In a recent microarray experiment (Christie & Eberl, unpublished), we have learned that all five of the CPAs are expressed in the Johnston s Organ and thus all five are potential partners to the V-ATPase in hearing. In the Malpighian tubule, other pumps localize to the basal membrane with the Na/K pump. These belong to the greater cation chloride cotransporter (CCC) superfamily. The CCCs divide into four subfamilies. Members of one subfamily use the Na + gradient set up by the Na/K pump to enhance K + transport into the principal cell. Mutations in members of this subfamily of CCCs reduce K + pumping in the Malpighian tubule (Arroyo et al, 2013; Sun et al, 2010). One of these genes, CG31547, is expressed in the Johnston s Organ, according to microarray results from our lab (Christie & Eberl, unpublished) and may be located on the ablumenal membrane of the scolopale cell alongside the Na/K pump. 9

22 Chapter II. Methods A. RNAi Knockdown The current work builds on work previously done on the Na/K pump (Roy et al, 2013). There, an RNAi knockdown approach was used with the chordotonal organ driver ato-gal4 and a scolopale cell specific driver, nompa-gal4. This approach first establishes the importance of the targeted gene in the Johnston s Organ in general, using ato-gal4, which expresses in the chordotonal sense organ precursor and its entire lineage. Then the nompa-gal4 driver establishes its specific role in the scolopale cell and suggests that it is involved in maintaining the receptor lymph. Two important concerns with the RNAi approach are the following. First, the RNAi construct may target genes other than the intended gene, so called off-target effects. To minimize this concern, particular care was taken in choosing RNAi constructs that do not have predicted off target effects. In the case of the V-ATPase, converging evidence from multiple subunits also discounts the possibility that the sum effects observed on knockdown are off-target effects. The situation is less clear with the cation chloride cotransporters and cation proton antiporters. The other concern with RNAi knockdown studies is the possibility of incomplete knockdown. Some mrnas can escape the knockdown machinery and enough can escape to mask the deficiency. Using the RNAi method also cannot detect any other systematic compensations the target tissue might make for the absence of the target protein. However, this method was preferred because of its ability to overcome developmental lethality and its ability to pinpoint specific cell types within a tissue. Here, it is important to establish the role of these ion systems in the maintenance of the receptor lymph. The RNAi technique allows studying anatomically targeted deficiencies without a global deficiency. 10

23 1. RNAi constructs for the V-ATPase The V-ATPase is a multisubunit complex. It is composed of twelve subunits and most of the subunits have multiple genes coding for them. Among these only the genes that are expressed in the Johnston s Organ were tested. These subunits are present in Table 1 below with their sources noted. Table 1 Stock center and stock number of RNAi construct flies used to target V-ATPase subunits Subunit Gene Stock Stock number Center a vha100-1 BDSC c vha16-1 BDSC d vhaac39-1 BDSC vham9.7-a Vienna KK e vham9.7-b Vienna GD30384 vham9.7-c BDSC A vha68-1 BDSC vha68-2 BDSC B vha55 BDSC C vha44 BDSC 3384 D vha36-1 Vienna KK vha36-3 Vienna KK E vha26 BDSC F vha14-1 Vienna KK

24 G vha13 BDSC H vhasfd BDSC AC45 vhaac45 BDSC VhaPRR vham8.9 Vienna GD RNAi constructs for cation proton antiporters There are five Drosophila cation proton antiporters (CPAs) genes. The microarray (Christie & Eberl, unpublished) showed that all five are expressed in the Johnston s Organ. RNAi constructs for all CPAs accept for Nha2 was obtained. At the time of reagent acquisition, no RNAi constructs for this antiporter was available. Table 2 contains the source of the RNAi stock. Table 2 Stock center and stock number of RNAi flies targeting CPA's Pump CG number Stock Stock number Center Nhe1 CG12178 BDSC Nhe2 CG9256 Vienna KK Nhe3 CG11328 Vienna KK Nha1 CG10806 BDSC

25 3. RNAi constructs for cation chloride cotransporters There are five Drosophila cation chloride cotransporters (CCCs) (Sun et al, 2010). Four of them were expressed in the Johnston s Organ, according to microarray experiments (Christie & Eberl, unpublished) and we studied three. Of the three we studied, one is thought to transport both Na + and K + along with Cl -. The other two CCCs are less clearly explored. The source of the three genes targeted via RNAi are listed in Table 3. Table 3 Stock center and stock number of RNAi flies targeting CCC's Gene Stock Center Stock number CG12773 Vienna KK CG10413 TRiP BDSC CG31547 Vienna KK Driver lines The two drivers used are ato-gal4 and nompa-gal4 (Roy, 2013). ato-gal4 (w 1118 ; ato- Gal4, UAS-Dicer2) drives expression in all cells of the Johnston s Organ. However, in adult tissue, ato-gal4 expression in the scolopale cell is low. Therefore, in cases where RNAi targets are specifically important in scolopale cells, the ato-gal4 driver may fail to produce a strong enough knock down. The other driver used is nompa-gal4 (nompa-gal4; UAS-Dicer2/CyO). nompa-gal4 drives expression specifically in the scolopale cell. 13

26 B. Electrophysiology 1. Rearing flies for electrophysiology The fact that the nompa-gal4 driver is located on the X chromosome was exploited. Crosses were designed so that the female offspring inherited the nompa-gal4 driver and the RNAi-knockdown construct. These flies were compared to their male siblings who only contained the RNAi-knockdown construct but not the driver, in an otherwise similar genetic background. For most of the genes (vha68-1, vha44, vha36-1, vha36-3, vha14-1, vha13, vham9.7-a, vham9.7-b, vham8.9, Nhe1, Nhe2, Nhe3, Nha1, CG12773, CG31547, and CG10413) the cross producing flies inheriting both the ato-gal4 driver and the respective knockdown construct was done at the same time as the nompa-gal4 cross and the same male fly from the nompa-gal4 cross inheriting the knockdown construct but none of the drivers was used as a control. For the rest of the genes (vha68-2, vha55, vha26, vhasfd, vha100-1, vha16-1, vhaac39-1, vham9.7-c and vhaac45) an age matched fly carrying the knockdown construct raised in a similar environment was used as a control. The studies done on the Na/K pump (Roy et al, 2013) used a fly inheriting the driver but not the knockdown construct as a control. Inheriting either of the two drivers does not influence the SEP. In contrast to that work, we preferred using a construct control, flies that inherit the knockdown construct but not the driver, because that would control for the effect of inheriting the knockdown construct. In this way, the only difference between knockdown flies and control flies is inheriting the driver, a difference that does not influence fly hearing. Flies were reared at 25 ºC at 12 hour light/dark cycle with 60-70% humidity. After sorting, flies were kept in an uncontrolled tabletop environment. Flies tested were older than one 14

27 day but younger than a week. A fly s ability to hear does not diminish until after a month (Christie & Eberl, unpublished). In addition to this fact, because age-matched controls were used, any differences in SEP observed between control and knockdown flies should be due to the knockdown, not a reflection of the fly s age. 2. Electrophysiology Flies older than a day but younger than a week were mounted on pipette tips and sound evoked potentials (SEPs) are recorded, as described by (Eberl & Kernan, 2011). Briefly, a computer generated component of the courtship song was routed to the fly s head from a speaker. The fly was positioned so that it is 1mm away from the end of the tube. Two tungsten electrodes were used, one placed on the antennal nerve, the other on the head as a reference. The amplitude of the antennal response to ten pulses was used from each antenna. The number of antenna recorded from is indicated per genotype in the text. The signal was amplified by a DAM50 differential amplifier (WPI) and digitized and normalized using Superscope II software (GW Instruments) (Roy et al, 2013). C. Immunohistology 1. Solutions Standard PBS and PBT solutions. 4% (w/ml) paraformaldehyde. 1mg/1ml Bovine serum albumin in PBT (BSA). 10%, 20% and 30% sucrose solutions in PBS. 2. Fixation and cryosectioning Antenna of flies up to a week old are dissected in PBT and fixed for 15 minutes in 4% paraformaldehyde, washed in PBT for 45 mins and incubated in 10% sucrose overnight at 4 C, then incubated in 20% and 30% for a day each at 4 C and embedded in OCT and cut at -20 C at 25 µm. Sections are preserved at -20 C. 15

28 3. Processing Sections are dried at room temperaturex washed with PBT for 1 hours, BSA for 3 hours, and then incubated overnight at 4 C with primary antibodies. Primary antibodies are washed with BSA and then secondary antibodies are applied for 2 hours and washed with BSA. Sections are counterstained with TexasRed Phalloidin for two hours and mounted with flouromountg and sealed with nail polish. 4. Antibodies Polyclonal rabbit anti-gfp conjugated with Alexa488 (1:200) recognizes EYS-GFP as well Nha2-YFP and was used to recognize all fusion proteins. Polyclonal primary rabbit antivha55 (1:1000) with antirabbit Alexa488 (1:200) was used to recognize V-ATPase subunit B. 22C10 monoclonal mouse antibody (1:20) with antimouse Alexa568 (1:200) was used to mark neurons as noted. Polyclonal rabbit antibodies were used against Nhe1, anti Nhe2 and anti Nhe3 to study their localization. The Nhe1, Nhe2, Nhe3 and anti vha55 antibodies were provided Julian Dow (Day et al, 2008). 5. Special flies EYS-GFP flies were provided by Andy Zelhoff and Nha1-YFP and Nha2-YFP flies were provided by Julian Dow. The two YFP fusion proteins were also used by Day et al (2008). 16

29 Chapter III. Vacuolar-ATPase The V-ATPase is the proton pump. Although it is abbreviated from Vacuolar-ATPase based on early studies in yeast, it is not specific to vacuoles. It can be present in most membranes: in the ER system, vacuoles, vesicles and the plasma membrane (Wieczorek et al, 1999). A. Domains of the V-ATPase complex The V-ATPase is a multisubunit complex. It is further complicated in higher eukaryotes by the fact that some subunits are encoded by multiple genes. When dealing with the V-ATPase complex using a gene specific knockdown approach, this merits special consideration. It is possible to divide the V-ATPase into two major complexes using a structural approach and a functional approach (Maxson & Grinstein, 2014). Traditionally, it has been divided into two structural multi-subunit domains: The membrane integral V0 domain and the cytoplasmic V1 domain. These two domains are depicted in Figure 3A. Subunits belonging to the V0 domain are numbered, in lower case letters, a through e, excluding b, and are generally involved with proton translocation and membrane specification. The V1 domain is generally involved in ATP hydrolysis and regulation. It is composed of subunits A through H, represented with capital letters. 17

30 Figure 3 The different components of the V-ATPase. The V-ATPase is a multisubunit complex that can be divided into two major domains in two different ways. A. divides the V- ATPase complex structurally in terms of each domain s relationship to the membrane. The V0 domain is membrane integral and contains subunits primarily involved in localization and proton extrusion. The V1 domain contains the cytoplasmic subunits primarily involved in ATP hydrolysis. B. divides the V-ATPase functionally into the peripheral stator and the central rotor. The peripheral stator anchors the V-ATPase and creates a reference for the rotor. The central rotor turns in response to ATP hydrolysis. In addition to this structural subdivision, the V-ATPase can also be broken down into two functional domains. This is depicted in Figure 3B. The two domains are the peripheral stator and the central rotor. The rotor is composed of the subunits that move in response to ATP hydrolysis. These include the subunits d, D, F, c, c`, c``. The rest of the subunits make up the stator, the subunits that do not turn with ATP hydrolysis. These are subunits A, B, C, E, F, G, H, a, and e. The original work done on the V-ATPase were done in yeast (Nelson, 2003). Yeast is a convenient model system for studying the V-ATPase because with only one exception all subunits are coded by a single gene in the yeast genome. The only exception to this rule is the stator subunit a, for which there are two genes. The product of one of the genes targets the 18

31 complex to the internal membranes and the other gene product targets the complex to the plasma membrane. B. V-ATPase subunits and genes expressed in the Johnston s Organ The original subunits defined in yeast were identified using the acidic phenotype. Yeast strains that have a defective copy of the genes producing the subunits can grow normally at a ph of 5.5 but cannot grow in medium at a neutral or basic ph because it fails to acidify internal compartments (Nelson, 2003). Additional subunits were identified in higher eukaryotes, such as the vhaac45 (accessory subunit, 45 kilodaltons) subunit and the vhaprr (prorenin receptor) subunit. Based on this initial work, it may be assumed that the entire complex fails to function in the absence of any individual subunit. In Drosophila, the system is immensely complex. Some subunits, especially those belonging to the V0 domain, are encoded by more than one gene. For example, the proton translocating subunits a and c each are coded by five different genes. Successfully knocking down one gene will conceivably knockdown the function of the V-ATPase on the membrane the targeted gene functions on. However, whether or not the other genes coding the same subunit but targeting other membranes of the same cell are capable of ameliorating the loss of the gene product of interest cannot be easily elucidated with a knockdown approach and is not within the scope of the present work. Our interest in the V-ATPase primarily stems from its involvement in ion homeostasis in the Malpighian tubule (Day et al, 2008; Xiang et al, 2012) where similar ion transport challenges exist as in the scolopidia. There it is localized with cation proton antiporters on the apical membrane of the principal cells. This is a system that maintains high K + concentrations in the principal cell cytoplasm as well as in the excreted fluid. In addition, the Na/K pump is located on 19

32 the basal membrane, bringing K + from the hemolymph into the principal cell itself. Thus the V- ATPase, with respect to the fact that it is located on a membrane opposite to that of the Na/K pump and with respect to the fact that it is located on a membrane separating a compartment high in K + ions from a compartment even higher in K + ions, is a reasonable candidate for ion maintenance on the lumenal membrane of the scolopale cell. Together with the fact that mutations in the B and a subunits of the V-ATPase were linked with sensorineural hearing loss in humans with distal renal tubular acidosis (Karet, 2002; Miura et al, 2013, Norgett et al, 2012), the involvement of the V-ATPase complex in the Johnston s Organ is worth investigating. C. Changes in SEP of flies with subunits knocked down with RNAi To this end, we identified the genes coding V-ATPase subunits expressed in the Johnston s Organ and then knocked them down individually. The genes encoding V-ATPase subunits expressed in the Johnston s Organ are shown in Figure 4. We used the ato-gal4 driver to express the RNAi constructs in all of the Johnston s Organ to reveal the general involvement of the subunit in facilitating hearing. We then used the nompa-gal4 driver to specify the scolopale cell and reveal the subunits role in maintaining the receptor lymph, the fluid in which mechanotransduction takes place. This is a paradigm previously used to study the function of the Na/K pump in the Johnston s Organ (Roy et al, 2013). Electrophysiology was done on the knockdown flies to check the antennal nerve s response to a component of the Drosophila courtship song (Eberl & Kernan, 2011). This is called the pulse song and has been well established as a reliable stimulus for measuring the fly s ability to hear. The amplitude of the antennal nerve s response is called the sound evoked potential (SEP). The results of these experiments are summarized in Table 4 below. Bar graphs of subunits that have significantly 20

33 different SEP from their construct controls are shown in Figure 5 for the ato-gal4 driver and Figure 6 for the nompa-gal4 driver. Figure 4 Genes encoding V-ATPase subunits expressed in the Johnston's Organ. Drosophila V-ATPase is complex in that some subunits are encoded by more than one gene. Because of our interest in the Johnston s Organ only subunits known to be expressed from a microarray experiment (Christie & Eberl, unpublished) are studied. All shown subunit genes were knocked down one by one using two different drivers summarized in the text. Table 4 All Drosophila genes encoding V-ATPase genes, their expression in the Johnston s Organ and the significance between knockdown flies and construct controls. All of the genes coding for Drosophila V-ATPase subunits are listed below. The genes that are not expressed in the Johnston s Organ were not studied and are shaded in grey. Of the genes that were studied, the genes that produced a significantly lower SEP are indicated with the respective driver. All knockdown possibilities not shaded in grey were studied but no statistical (two-tailed Student s t-test) differences were observed between knockdown flies and their construct controls. Subunit Gene In the Johnston s Organ vha68-1 Yes ato-gal4 nompa-gal4 A vha68-2 Yes P<.05 vha68-3 No 21

34 B vha55 Yes C vha44 Yes vha36-1 Yes P<0.05 P<0.01 D vha36-2 vha36-3 No Yes E vha26 Yes P<0.05 P<0.01 F vha14-1 Yes P<0.05 vha14-2 No G vha13 Yes H vhasfd Yes vha100-1 Yes P<0.01 a vha100-2 vha100-3 vha100-4 vha100-5 Yes No No No vha16-1 Yes P<0.05 vha16-2 No c vha16-3 No vha16-4 No vha16-5 No d vhaac39-1 Yes 3 rd antennal segment falls off P<0.01 vhaac39-2 No 22

35 e vham9.7-a vham9.7-b vham9.7-c vham9.7-d Yes Yes Yes No AC45 vhaac45 Yes P<0.05 P<0.01 PRR vham8.9 Yes SEP (µv) Figure 5 Subunits coded by vhaac45, vha68-2, vha36-1, vha26, vha14-1 and vha16-1 show significantly reduced SEPs when knocked down with ato-gal4. (vhaac45, N=14, vha68-2, N=13, vha36-1, N=14, vha26, N=12, vha14-1, N=14, vha16-1, N=16-1). The subunits shown demonstrate a decrease in the Sound Evoked Potential in the auditory nerve. They are significantly different (p<0.01 for all except vha16-1 for which p<0.05) from their controls with a two-tailed Student s t-test. Error bars represent standard error. We found that knocking down subunit a and subunit F with the ato-gal4 and knocking down subunit c with the nompa-gal4 driver significantly reduces the SEP of flies. The same reduction was observed using both drivers for one isoform of subunit D (vha36-1), subunit E, AC45 and subunit d. 23

36 Figure 6 Scolopale cell specific knockdown of V-ATPase subunit genes produces significant reductions in SEP with vhaac45, vha36-1, vha26, vha100-1 and vhaac39-1. (vhaac45, N=14, vha36-1, N=14, vha26, N=14, vha100-1, N=13, vhaac39-1, N=7). The subunits shown when knocked down with nompa-gal4, are statistically different from their sibling controls by a two-tailed Student s t-test analysis. Error bars are standard error Subunits a and c are directly involved in proton translocation. Notice that with knockdown, both produced a significant decrease in the SEP with a driver. Knocking down subunit a with nompa-gal4 produced a more noticeable effect then knocking down with ato- Gal4. This appears to be a discrepancy because both ato-gal4 and nompa-gal4 are expressed in the scolopale cell. However, ato-gal4 drives expression early in development. In adult tissue, nompa-gal4 expression is stronger in the scolopale cell than ato-gal4 is. Therefore, especially in systems that are pronounced in the scolopale cell, nompa-gal4 may reveal an effect when ato- Gal4 fails to. With subunit a, this appears to be the case. Only one of the five subunit c genes is expressed in the Johnston s Organ. Knocking this subunit down with ato-gal4 succeeds in revealing its importance, while nompa-gal4 fails to produce a similar effect. 24

37 Furthermore, certain interesting phenotypes were observed. Knocking down the vhaac39-1, the d subunit, with nompa-gal4 produced near lethality, with very few progeny emerging as adults. Knocking this subunit down with ato-gal4 produced flies that were born with a decaying antenna. Their antennae fall off within hours after eclosing. These flies are by definition deaf. Recall that Drosophila hearing begins with the feather-like arista moving in response to a sound wave. When the segment of the antenna containing the arista falls off, the fly cannot hear. Knocking down the gene vha68-2, encoding subunit A, with ato-gal4 produced a similar decaying phenotype. These flies had rigid antennae. The fact that they have significantly reduced SEPs may be a product of the inability of the antenna to move rather than a defect in ion homeostasis of the receptor lymph. The observed reduction of the SEPs suggests an important role for the proton gradient in mediating hearing. D. Locating the V-ATPase in the Johnston s Organ It is clear that the proton gradient plays a role in hearing. It also seems that this gradient is particularly important in the scolopale cell. This lends support to the idea that it is involved in receptor lymph maintenance. This produces the hypothesis that it is located on the lumenal membrane of the scolopale cell. That kind of staining would look like a thin ring of the antibody slightly internal to the scolopale rods, which form a tight ring around the lumenal membrane. Staining of Canton-S antennae with antibodies against the B subunit Figure 8 shows that this is not the case. Although there is general staining in the cytoplasm of the neuron, reflecting the fact that the V-ATPase often coats vesicles, it does not appear to be specifically located on the lumenal membrane of the scolopale cell. Instead, it stains specifically at the ciliary dilation level in the scolopale space. Figure 9 shows a cross-section of the scolopidia stained for V- 25

38 ATPase subunit B. Notice that the staining for the V-ATPase fills the scolopale space between the scolopale rods rather than forming a thin ring where the lumenal membrane would be. TexasRed Phalloidin and V-ATPase staining do not overlap. However, because the lumenal membrane is immediately internal to the scolopale rods, some level of overlap would be expected if the V-ATPase was located on the lumenal membrane. Figure 7 Canton-S antenna stained with anti vha55 (green) and TexasRed Phalloidin (red). Canton-S tissue for the product of vha55 (V- ATPase subunit B) shows extensive staining in the cell bodies of the neurons, consistent with the fact that the gene product of vha55 is cytoplasmic. Antibodies against the product of vha55 also stain extensively at the proximal end of the scolopale rods (counter stained with TexasRed Phalloidin and shown in red) at the level of the basal bodies as well as more distally in the scolopale space at the level of the ciliary dilation. Figure 8 Cross section of Canton-S stained with anti-vha55 gene product (green) and TexasRed Phalloidin (red). Antibodies against the gene product of vha55 (subunit B) and counterstained with TexasRed Phalloidin show that the V-ATPase is located within the scolopale space. This is because TexasRed Phalloidin stains actin filaments in the scolopale rods that mark the edges of the scolopale space. The staining for subunit B is contained within the circle formed by the scolopale rods. 26

39 The localization of the V-ATPase resembles that of Eyeshut (EYS), a protein involved in osmoregulation that also decorates the scolopale space (Cook et al, 2008). Antennal tissue expressing UAS-EYS-GFP with the nompa-gal4 driver is shown in Figure 10. Exactly as seen with the V-ATPase, EYS is located basally in the scolopidium slightly above the basal bodies. It also stains brightly at the level of the ciliary dilation. To the author s knowledge there are no known interactions between EYS and the V-ATPase. Figure 9 EYS-GFP fusion with nompa-gal4 driver (green), 22C10 (blue), TexasRed Phalloidin (red). EYS localizes in the scolopale space level with the ciliary dilation. EYS also localizes more proximally at the base of the scolopale space. When 22C10 stains the membrane of the neurons. Notice how the EYS staining combined with 22C10 is remarkably similar to subunit B staining in Figure 7. 27

40 Chapter IV. Secondary Transport Thus far, we have discussed the role of the Na/K pump in the Johnston s Organ (Roy et al, 2013). In the previous chapter, we investigated the V-ATPase based on its relationship to the Na/K pump in the Malpighian tubule and considered it as a candidate energizer in the lumenal membrane of the scolopale cell to pump K + ions from the cytoplasm of the scolopale cell into the receptor lymph, which is thought to have even higher K + concentrations. These pumps constitute the primary ion transport mechanisms. Primary transport uses the energy of ATP hydrolysis to produce an ion gradient. Both the Na/K pump and the V-ATPase use ATP hydrolysis to produce Na + and proton gradients respectively across the membrane they are located on. In contrast to primary transport, secondary transport uses an already present gradient (usually maintained by primary transport) to pump other ions and solutes across the membrane. Secondary transport pumps are often colocalized or even coupled with the respective primary transport. In this chapter, we will investigate the classic partners to the V-ATPase, cation proton antiporters (CPAs). CPAs take advantage of the proton gradient established by the V-ATPase to pump other ions like Na + and K + back across the membrane. We will then discuss the possible role of the cation chloride cotransporters (CCCs) which are partners to the Na/K pump on the basal membrane of the principal cells in the Malpighian tubule. A. Cation proton antiporters (CPAs) There are five CPA s in the Drosophila genome. These are often divided into two groups. The first group includes the relatively well-studied three Na/H exchangers, Nhe1, Nhe2 and Nhe3. These have well-established homologs in the mammalian genome and have been well 28

41 studied in humans and mice. These are thought to exchange Na + specifically by taking advantage of a proton gradient. Two more ancient antiporters have been discovered, the Na/H antiporters Nha1 and Nha2, which are thought to also be able to transport K + (Day et al, 2008). All five of the genes encoding these pumps are expressed in the Johnston s Organ, according to a microarray experiment (Christie & Eberl, unpublished). We studied four of them, Nhe1, Nhe2, Nhe3 and Nha1, for which RNAi lines were available. We used the same general approach as described in chapter III. As shown in Figure 10, of these four, Nhe3 produced a significant reduction in the SEP using nompa-gal4. As noted earlier, when the target is important specifically in the scolopale cell, because of the fact that it is not as strongly expressed in the scolopale cell as nompa-gal4, ato-gal4 may fail to reveal the importance of the targeted gene in ion maintenance. Because of this, it seems that Nhe3 may be a partner to the V-ATPase in the scolopale cell. Figure 10 Nhe3 knockdown with nompa-gal4 produces significantly reduced SEPs. (Nhe1, N=14, Nhe2, N=16, Nhe3, N=16, Nha1, N=14). Knocking down CPA s in the Johnston s Organ does not produce immediately meaningful observations. The only CPA that shows statistically significant (two-tailed Student s t-test, p<0.01) difference from its construct control is Nhe3 when knockdown specifically in the scolopale cell. This indicates that Nhe3 is a candidate exchanger on the lumenal membrane of the scolopale cell and may be a partner to the V-ATPase. 29

42 We have antibodies against Nhe1, Nhe2 and Nhe3 produced in rabbit (Day et al, 2008). Experiments to reveal the location of these pumps in the scolopidium are in progress. We also have YFP fusion proteins with Nha1 and Nha2. Although electrophysiology was not done on Nha2, for which we have no RNAi lines, localization studies may shed light on this antiporter. Figure 11 Nha2-YFP fusion (green) expressed with ato-gal4. Counterstained with TexasRed Phalloidin (Red). Actin-rich scolopale rods line the lumenal membrane of the scolopale cell and are visualized with TexasRed Phalloidin. Note that ato-gal4 driven Nha2-YFP expression does not show in the scolopale cell level of the scolopidium. The only place that Nha2-YFP appers is at the neuronal level when expressed with ato-gal4. Nha2 driven with ato-gal4 stains in Johnston s Organ neurons. The fusion construct expressed with nompa-gal4 stains in a manner reminiscent of EYS. The antigfp Alexa488 conjugated antibody was used to recognize the YFP tag in this circumstance. The same antibody was used to study EYS localization by targeting a EYS-GFP fusion. Work must be done to determine if the apparent dilation level specific localization of these two may be a result of the antibody being used. 30

43 Note, the same antibody was used to stain Nha2-YFP fusion when driven by ato-gal4. The ciliary dilation level staining seen with the nompa-gal4 driver was not seen there, indicating that the green staining in Figure 12 is not an artifact. Figure 12 Nha2-YFP fusion (green) expressed with nompa-gal4. Counterstained with TexasRed Phalloidin. Scolopale cell-specific expression of an Nha2-YFP fusion protein shows that Nha2 may be located distally to the ciliary dilation in the scolopale space. This suggests that the scolopale cell may be more involved with decorating the scolopale space than previously thought. 31

44 B. Cation chloride cotransporters (CCCs) Figure 13 The CCC family tree. There are five Drosophila CCCs shown here in bolded text. The nine vertebrate CCCs are boxed in grey. Notice that the five Drosophila CCCs represent the four subgroups, shown in color code (modified from Sun et al, 2010). Cation chloride cotransporters (CCCs) have only recently been studied in Drosophila. They make up Solute Carrier Family 12. This family uses other gradients to electroneutrally transport chloride. There are four subfamilies: 1) CCCs that use the Na + gradient created by the Na/K pump to move Na +, K +, and Cl - into the cell at a 1:1:2 ratio. These are the NCCs and NKCCs. In Drosophila, there are two genes belonging to this subfamily, CG4357 and CG31547 (sometimes called Ncc69 and Ncc83 respectively). 2) CCCs that pump K + independently from the Na + gradient. These are the KCCs. They electroneutrally move K + and Cl - out of the cell at a 32

45 1:1 ratio. In Drosophila, this subfamily is represented by CG ) CIP type, represented by CG in Drosophila and 4) CCC9 type, represented by CG Not much is known about the latter two types. This is reviewed by Sun et al (2010) and Hartmann and Nothwang (2015). CCC function in neurons is extremely important. The change in the ratio of NKCC to KCC is cited as the reason behind the fact that developmentally excitatory GABAergic neurons become inhibitory in adult brains. This is reviewed in Kahle et al (2008). NKCC1 null mice show inner ear dysfunction (reviewed by Delpire & Mount, 2002). They have a collapsed endolymphatic cavity because of the failure to maintain the endolymph. However, our primary interest in CCCs stems from a study performed in the Malpighian tubule, where it located on the basal membrane of the principal cell alongside the Na/K pump (Rodan et al, 2012). The inhibition of the Na/K pump with ouabain reduces, but does not abolish, K + secretion, indicating that there are other pumps that mediate K + uptake. Furthermore, mutations in the CG4357 gene encoding Ncc69 also have reduced K + secretion. Most importantly, administering oaubain in Ncc69 mutants, does not further reduce K + secretion, meaning that the Na/K pump primarily functions to power the CCC. In contrast to this finding, knockdown of Na/K pump subunits in the Johnston s Organ leads to deafness (Roy et al, 2013). This suggests that the Na/K pump plays a bigger role in the Johnston s Organ than in the Malpighian tubule and that the role of the CCCs may not be as critical. However, one important distinction must be made between these two structures. Fluid in the Malpighian tubule lumen is constantly drained out. As opposed to that, the scolopale space is a sealed environment with minor changes in ion concentrations with the opening of ion channels. In light of this, the role of CCCs may become more important in the presence of stressors. 33

46 Because of the Na/K pump s role in the lumenal membrane of the scolopale, we also explored CCCs as secondary transport. There are five CCC genes in Drosophila. Two of them belong to the Na dependent CCCs (CG3157 and CG4357). Because CG4357 was not expressed in the Johnston s Organ, according to a microarray study in the lab (Christie and Eberl, unpublished) it was not studied. There is one Drosophila CCC in each of the remaining categories and these are expressed in the Johnston s Organ: CG5594, a Na + independent CCC, CG10413, a CIP type, and CG12773, a CCC9 type. We knocked down CG3157, CG10413 and CG12773 with ato-gal4 and nompa-gal4 using the RNAi approach previously described. Figure 14 Knocking down CCC genes does not change the SEP. (CG31547, N=12, CG12773, N=20 and CG10413, N=14). Knocking down CCC genes with the two drivers does not reduce the SEP. The differences are assessed with a two-tailed Student s t-test. The significant difference between CG31547 knocked down with ato-gal4 (p<0.01). We would expect that if one of these transporters is required in the Johnston s Organ for hearing, then RNAi knockdown of its genes should result in a significant reduction in the SEP. However, our experiments do not show this (Figure 14). RNAi knockdown of CG12773 and CG10413 do not produce a significant change in the SEP. ato-gal4 knockdown of CG

47 resulted in a statistically significant increase. There is not an easily explained observation. It may be a Type 1 statistical error. This is a type of error that occurs when an observation is statistically significant due to chance. However, alternative explanations will be raised in the Discussion section. Ultimately, more experiments are needed to distinguish the possibilities. 35

48 Chapter V. Discussion Because of the way the scolopale cell wraps around the dendrite of the neuron, it creates three fluid filled compartments. Its cytoplasm separates the hemolymph from the receptor lymph. To achieve a receptor lymph high in K + concentrations, K + ions must pass through both of the membranes of the scolopale cell: the ablumenal membrane forming the boundary between the hemolymph and the scolopale cell cytoplasm, and the lumenal membrane forming the walls of the scolopale space. The Na/K pump is usually thought of as the main pump establishing Na/K gradients across cellular membranes. However, it is not the Na/K pump directly maintaining either the receptor lymph in Drosophila (Roy et al 2013) or the endolymph in mammals. In spite of this knocking down Johnston s Organ specific subunits of the Na/K pump using RNAi technology does in fact result in deafness in flies (Roy et al, 2013). However, the Na/K pump is not located on the membrane of the scolopale cell lining the receptor lymph. It is instead on the ablumenal membrane of the scolopidium on the boundary between the scolopale cell cytoplasm and general bodily fluid, the hemolymph. In the Malpighian tubule, the proton pump, the V-ATPase, is located on a membrane opposing the Na/K pump and acts with secondary transporters to create a K + -rich lumen (Day et al, 2008). Based on this, it is expected that V-ATPase would be located on the lumenal membrane and function with secondary transporters, such as CPAs to maintain a K + -rich receptor lymph. We would also expect secondary transport partners to the Na/K pump, for example CCCs, to be located alongside the Na/K pump on the ablumenal membrane (Rodan et al, 2012). 36

49 Figure 15 The present model of ion transport in the scolopidium Here, we have demonstrated with tissue specific RNAi mediated knockdown of individual subunits of the V-ATPase expressed in the Johnston s Organ according to an unpublished microarray experiment (Christie & Eberl, unpublished), that the proton gradient is important in mediating hearing. More than one subunit involved in directly producing the proton gradient, when knocked down individually, produced a reduction in the antennal response to a sound stimulus. However, none of these flies were completely deaf. It is not clear if the lack of absolute deafness is a result of the drawbacks of RNAi technology, in that the targeted genes may not have been completely knocked down, or if the proton gradient is important in maximal response of the antennal nerve. One important consideration must be put on the subunit e of the V-ATPase. Three of the five Drosophila genes coding for this subunit were expressed in the Johnston s Organ. The three 37