Protein Trap Lines of Drosophila to demonstrate Spatio-Temporal Localization of Proteins in an Undergraduate Lab

The objective of this teaching note is to generate a laboratory exercise, which allows students to get a hands-on experience of a cell biology technique. The short duration of the laboratory classes is the biggest challenge with the development of a cell biology lab for an undergraduate curriculum. Therefore, it is necessary to design a laboratory exercise that enables the students to carry out cell biological assays in the desired time. This laboratory exercise focuses on tracking protein expression levels along a spatial (space) and temporal (time) axis in developing Drosophila melanogaster organ primordium. Here we use the protein trap model developed in Drosophila to demonstrate the sub-cellular localization of proteins. The protein trap transgenic flies have green fluorescent protein (GFP) reporter tags to the full-length endogenous proteins that allow observation of their cellular as well as sub-cellular distribution. Since the life cycle of Drosophila is short, it is easy to rear them in the lab and also use them as an excellent model for an undergraduate lab curriculum. The goal of this exercise is to train undergraduate students and teach them the use of one such powerful tool which enables the localization of proteins

GMR42H01

dve enhancer 48150 ID - GMR42H01 Location - 2R: 18158419, 18162751 Base pairs - 433

GMR35A04

dve enhancer ID - GMR35A04 Location - 2R: 18125755, 18128365 Base pairs - 261

GMR41F05

dve enhancer 50133 ID - GMR41F05 Location - 2R: 18139042, 18142672 Base pairs - 363

GMR41C04

dve enhancer ID - GMR41C04 Location -2R: 18143594, 18147064 Base pairs - 347

Shop Talk: Annual Drosophila Research Conference, 2010

This year, the Genetics Society of America (GSA) received a record-breaking number of registrants to the conference. Despite low attendance at other scientific meetings, according to GSA Meetings Manager, Suzy Brown, this year’s conference had the ‘‘largest number of registrants than any other previous years.’’ There were 170 talks, more than 850 posters and 13 workshops; so there was a range of information that people could pick according to their interests

Drosophila Adult Eye Model to Teach Scanning Electron Microscopy in an Undergraduate Cell Biology Laboratory

We have devised an undergraduate laboratory exercise to study tissue morphology using fruit fly, Drosophila melanogaster, as the model organism. Drosophila can be reared in a cost effective manner in a short period of time. This experiment was a part of the undergraduate curriculum of the cell biology laboratory course aimed to demonstrate the use of scanning electron microscopy (SEM) technique to study the morphology of adult eye of Drosophila. The adult eye of Drosophila is a compound eye, which comprises of 800 unit eyes, and serves as an excellent model for SEM studies. We used flies that were mutant for lobe (L), eyeless (ey), and pannier (pnr) for our studies. The mutant flies exhibit different morphologies of the adult eye. We employed a modified protocol, which reduces sample preparation steps and makes it practically feasible to complete the protocol in assigned time for the cell biology laboratory. The idea of this laboratory exercise is to: (a) familiarize students with the underlying principles of scanning electron microscopy and its application to diverse areas of research, (b) to enable students to sharpen their observation and quantitative microscopy skills, and (c) minimize the preparation time for the instructor

Novel Neuroprotective Function of Apical-Basal Polarity GeneCrumbs in Amyloid Beta 42 (Aβ42) Mediated Neurodegeneration

Alzheimer\u27s disease (AD, OMIM: 104300), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). We have developed a transgenic Drosophilaeye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb) as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. We demonstrate a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration

Drosophila Eye Model to Study Neuroprotective Role of CREB Binding Protein (CBP) in Alzheimer’s Disease

Background: The progressive neurodegenerative disorder Alzheimer’s disease (AD) manifests as loss of cognitive functions, and finally leads to death of the affected individual. AD may result from accumulation of amyloid plaques. These amyloid plaques comprising of amyloid-beta 42 (Aβ42) polypeptides results from the improper cleavage of amyloid precursor protein (APP) in the brain. The Aβ42 plaques have been shown to disrupt the normal cellular processes and thereby trigger abnormal signaling which results in the death of neurons. However, the molecular-genetic mechanism(s) responsible for Aβ42 mediated neurodegeneration is yet to be fully understood. Methodology/Principal Findings: We have utilized Gal4/UAS system to develop a transgenic fruit fly model for Aβ42 mediated neurodegeneration. Targeted misexpression of human Aβ42 in the differentiating photoreceptor neurons of the developing eye of transgenic fly triggers neurodegeneration. This progressive neurodegenerative phenotype resembles Alzheimer’s like neuropathology. We identified a histone acetylase, CREB Binding Protein (CBP), as a genetic modifier of Aβ42 mediated neurodegeneration. Targeted misexpression of CBP along with Aβ42 in the differentiating retina can significantly rescue neurodegeneration. We found that gain-of-function of CBP rescues Aβ42 mediated neurodegeneration by blocking cell death. Misexpression of Aβ42 affects the targeting of axons from retina to the brain but misexpression of full length CBP along with Aβ42 can restore this defect. The CBP protein has multiple domains and is known to interact with many different proteins. Our structure function analysis using truncated constructs lacking one or more domains of CBP protein, in transgenic flies revealed that Bromo, HAT and polyglutamine (BHQ) domains together are required for the neuroprotective function of CBP. This BHQ domain of CBP has not been attributed to promote survival in any other neurodegenerative disorders. Conclusions/Significance: We have identified CBP as a genetic modifier of Aβ42 mediated neurodegeneration. Furthermore, we have identified BHQ domain of CBP is responsible for its neuroprotective function. These studies may have significant bearing on our understanding of genetic basis of AD

A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration

Alzheimer\u27s disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a Drosophila eye model of AD by targeted misexpression of human Aβ42 in the differentiating retinal neurons, where an accumulation of Aβ42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aβ42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aβ42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aβ42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aβ42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aβ42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aβ42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aβ42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aβ42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aβ42-mediated neurodegeneration