MSc Exit Seminar
Friday, May 11th, 2018 at 10:10am Earth Sciences Building, Room 3087
Van Phan (Yoshioka Lab)
Evaluation of GCaMP3 Protoplasts for the Analysis of Ca2+ Signaling in Plant Immunity
Cytoplasmic calcium [Ca2+]cyt elevation is an early event that occurs after recognition of various environmental stimuli. Utilization of Ca2+visualization tools such as genetically-encoded Ca2+ indicators has advanced our knowledge of the temporal and spatial nature of Ca2+ signals. GCaMP3 is a green fluorescence protein (GFP)-based genetically-encoded indicator that can be detected by a conventional fluorescence microscope and a plate reader system. In this study, the use of GCaMP3 to study plant Ca2+ signals in protoplasts was evaluated.
Firstly, Arabidopsis thaliana leaf mesophyll protoplasts derived from stable transgenic lines expressing GCaMP3 were evaluated in comparison to leaf discs. Using fluorescence microscopy and a plate reader, Ca2+ signals upon abiotic and biotic stimuli in protoplasts were assessed in a quantitative and qualitative manner. Ca2+signals upon various stimuli were detected by both microscope and a plate reader. Ca2+ signals detected in protoplasts were generally quicker and stronger than those in leaf discs. However, the signal patterns were fundamentally similar between protoplasts and leaf discs. Thus, it was concluded that protoplasts expressing GCaMP3 can be utilized to study Ca2+ signaling.
Next, the usage of the transient expression of GCaMP3 in protoplasts (by transfection) was tested. This can be a powerful tool to analyze Ca2+signals in various mutants. Several methods to detect Ca2+signals from GCaMP3-transfected protoplasts were evaluated utilizing the bacterial elicitor, flg22. However, the detection by a plate reader was not successful, likely due to the insufficient number of the cells expression GCaMP3 by transfection, while some signals could be observed in individual protoplasts under the microscope.
Taken together, protoplasts can be used for investigating Ca2+signals upon various stimuli using GCaMP3. However, further optimization of the protocol for transfection is required.
MSc Exit Seminar
Friday, January 12th, 2018 at 10:10am Ramsay Wright Building, Room 432
Syed Saad Husainie (Godt Lab)
Analysis of the Function of Protein Kinase C δ in Regulating Collective Cell Migration during Drosophila Oogenesis through Live Imaging
Border cell cluster (BCC) migration during Drosophila oogenesis has provided an excellent model to study collective cell migration. We have previously demonstrated that the serine/threonine kinase Protein Kinase C δ (PKCδ) is a regulator of BCC motility and protrusion morphology. My project aimed at examining the effects of altered PKCδ expression on BCC motility through live imaging, focusing mainly on BCC migration behaviour and protrusion dynamics. Although known for its pro-apoptotic role, I found no evidence of cell death in BCCs with increased or decreased PKCδ expression. Live imaging revealed that BCCs require normal levels of PKCδ expression to migrate at a normal speed. Increasing PKCδ expression levels in BCCs delayed detachment from the epithelium and significantly slowed down migration. Decreased PKCδ expression caused BCCs to migrate slower and less linearly during the early phase of migration. In addition, PKCδ-deficient BCCs showed defects in cellular protrusion dynamics, including frequent bifurcations and multiple cycles of extension and partial retraction. In summary, my analysis contributed to a better understanding of the function of PKCδ in regulating migration and protrusion dynamics during collective cell migration.
MSc Exit Seminar
Wednesday, December 13th, 9:10am –University of Toronto at Mississauga – CC-2150
Gordana Scepanovic (Stewart lab)
“Analysis of Drosophila Nervous System Development Following a Brief Exposure to Ethanol”
Improper neuronal mapping predisposes many of the neurodevelopmental deficits that arise in patients of Fetal Alcohol Spectrum Disorders (FASD). Here I used Drosophila embryos as a model to investigate the neuronal defects that arise after exposure to ethanol. Drosophila is an excellent model for studying the mechanisms underlying FASD due to their vulnerability to the toxic effects of alcohol and the ease of manipulating their development. Using this model, I was able to provide detailed morphological analyses of the effects of a brief ethanol exposure on Drosophila neural development. I also examined the mechanisms that may regulate the FASD phenotype, specifically the serum response factor (SRF) signaling pathway. SRF signaling regulates numerous functions in the nervous system, including neuronal growth and migration. Through SRF manipulation in Drosophila, I was able to show that SRF is necessary for proper Drosophila axonogenesis, and that it may contribute to the FASD phenotype.