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2014 Research Showcase
CADB Abstracts
Advisor : JEFF OMENS
Abstract Title : Sarcomere Length and Lattice Spacing of Myosin-Actin Filaments in Cardiac Models
Abstract : With the continual improvement of healthcare technology and services today, the population of the elderly increases year after year. This evidently leads to a high demand for better treatment of diseases that afflict the elderly population, such as heart disease. The effects of heart disease and heart failure can be best explained and examined at the intracellular level through the analysis and understanding of changes in heart cells [1]. The aim of this research project is to correlate acute changes in sarcomere structure to underlying substrate deformation in cardiac myocytes that are cultured on a deformable substrate. We expect that by stretching a substrate to which cardiac myocytes are attached, the cells will undergo the same amount of stretch, and consequently the sarcomere length and transverse lattice will show corresponding magnitudes of deformation. Similarly, by applying biaxial strain with a greater magnitude of deformation in the transverse direction compared to the longitudinal axis, we expect sarcomere length to remain unchanged but lattice spacing to increase slightly due to the conservation of cell volume. This regional difference in sarcomere dimension is expected to be a result of the non-uniform nature of the applied stress to the cell. References 1. Holubarsch C, Ruf T, Goldstein D, Ashton R, Nickl W, Pieske B, Pioch K, Lu¨demann J, Wiesner S, Hasenfuss G, Posival H, Just H, Burkhoff D. (1996). Existence of the Frank-Starling Mechanism in the Failing Human Heart. Investigations on the Organ, Tissue, and Sarcomere Levels. Circulation 94.4.683.
Abstract Title : Localization and trafficking of ATP-binding cassette transporters in sea urchin embryos
Abstract : ATP-binding cassette (ABC) transporters are conserved membrane-spanning proteins that efflux structurally diverse compounds. Differences in primary structure account for some variability in cellular function, but it is not fully understood how localization and trafficking, which dictate subcellular distribution, affect transporter function. The goal of this project was to determine mechanisms governing trafficking of ubiquitous ABC transporters in the blastula stage of Strongylocentrotus purpuratus embryos. I hypothesized that apically-localized ABC transporters would utilize trafficking pathways associated with apical recycling endosomes, whereas non-apical transporters would not. I co-expressed fluorescently tagged ABC transporters and Rab11-GTPase, a marker of apical recycling endosomes, via microinjection of embryos with synthetic mRNAs. Using confocal microscopy, I visualized the trafficking of newly synthesized transporters to the plasma membrane after inhibiting anterograde ER-Golgi membrane traffic with brefeldin-A. Previous reports indicate Rab11 is required for localization of ABCB1a to apical membranes in fertilized sea urchin eggs, but my preliminary results demonstrate that in blastulae, Rab11-positive vesicles do not primarily co-localize with the apical transporters ABCB1a, ABCB4, or ABCG2a, and that other mechanisms, such as direct streaming from the trans-Golgi network, are responsible for the trafficking of these apical transporters, although Rab11 may play a minor role. The basolateral transporter, ABCC1β, however, did demonstrate co-localization with Rab11 vesicles at apical and, surprisingly, lateral surfaces, indicating it may be involved in transcytosis, since canonically, Rab11 is only associated with apical vesicles. Continued testing with markers for trans-Golgi network and other trafficking pathways will identify the routes used to transport these proteins to the plasma membrane.
Advisor : DR. AMY KIGER
Abstract Title : Identification of Rab GTPase roles in muscle cell Transverse Tubule formation and remodeling
Abstract : Muscle cells rely on increases in cytoplasmic calcium levels for successful contraction. Transverse (T)-tubules are a network of tube-like invaginations from the plasma membrane that mediate this process by spreading depolarizing signals to activate calcium release from the sarcoplasmic reticulum. While the physiological significance of T-tubules is well understood, the mechanisms behind their formation and remodeling are unknown. We discovered that during Drosophila metamorphosis, the T-tubule network in abdominal muscle cells undergoes remodeling, as characterized by disassembly during the larval-pupal transition and rapid reassembly in the late pupal stage. Additionally, we have described specific roles in this T-tubule remodeling program for several genes involved in endosomal trafficking and with homologs associated with human myopathy. Thus, flies provide an ideal system to study the mechanisms underlying T-tubule structure. To identify additional T-tubule regulators, I screened the Rab GTPases for T-tubule localization and required functions. The Rabs are a protein family with diverse roles in coordinating cellular membrane trafficking processes. We speculated that one or more of the Rabs likely are involved in T-tubule development. Using muscle-targeted RNAi knockdown and expression of fluorescently-tagged Rab proteins, I identified several candidates. Most notably, I demonstrated that Rab35 localizes along T-tubules and is also required for T-tubule reassembly during pupal muscle cell remodeling. Rab35 has been implicated in endocytic recycling, further pointing to a possible pathway framework and significance of regulated endosomal trafficking in T-tubule remodeling. Importantly, our findings can enhance knowledge of how Rab mutations may be linked to human myopathy and heart disease.
Abstract Title : Neonatal Enteropathogenic Escherichia coli Infection Causes Long Term Behavioral Defects And Long Term Alterations In The Microbiota.
Abstract : Increasing evidence suggests that the intestinal microbiome exerts a major influence on mental health and behavior. In humans, repeated exposures to enteric bacterial infections that cause diarrheal disease in early life are associated with cognitive defects in adulthood, despite resolution of diarrhea with oral rehydration solution (ORS) treatment. Previous work from our lab demonstrated that cognitive defects caused by the enteric pathogen Citrobacter rodentium were stress?induced. Currently, there is no effective model that emulates the cognitive defects in adulthood associated with early life diarrheal diseases. Here we propose to establish a model of neonatal infection to study adult behavioral changes. Mice infected with enteropathogenic Escherichia coli (EPEC) at post-natal day 7 were found to exhibit baseline memory defects in adulthood but did not exhibit anxiety-like behavior when compared to sham infected neonates. qPCR analysis of fecal samples from adult, previously EPEC-infected, mice demonstrated long term changes in the composition of the microbiota compared to uninfected controls. Significant decreases were observed in segmented filamentous bacteria (SFB), E. rectale, and Bacteroides populations in EPEC inoculated mice relative to sham mice, whereas the population of Enterobacteriaceae increased. Taken together, these results demonstrate that early-life infection with enteric bacterial pathogens can have detrimental cognitive effects as well as causing long-term dysbiosis in mice. We speculate that the changes in the microbiota may underlie long-term post-infectious behavioral defects.
Abstract Title : Androgen Receptor as a Novel Co-regulator of Autophagy
Abstract : Autophagy is the cellular process by which proteins, macromolecules, and organelles are degraded though vesicular packaging and targeting to the lysosome. Transcription factor EB (TFEB) has been recently identified as a master regulator of lysosomal biology and autophagy. Not a lot is known about the regulation of TFEB. We have identified the Androgen Receptor (AR) as a new co-activator of TFEB. Using luciferase reporter assays and quantitative RTPCR, we show significant effects of AR levels on TFEB activity. Co-immunoprecipitation studies show an interaction of TFEB and AR. This suggests AR to be a novel autophagy regulator through its interaction with TFEB.
Abstract Title : Pdgfr signaling is required for specification of hematopoietic stem cells
Abstract : Hematopoietic stem cells (HSCs) are responsible for the replenishment of all adult blood cells throughout the lifespan of an individual. For this reason they are extremely useful in clinic for the treatment of a multitude of hematological abnormalities such as leukemia or lymphoma, however the need for immune matching between patient and donor bone marrow is a major limitation. Thus a current research focus for regenerative medicine and stem cell biology is to establish transplantable hematopoietic stem cells from embryonic stem cells or induced pluripotent cells (iPSCs) in order to treat patients, however this feat has not been achieved and indicates that our understanding for the molecular mechanisms involved in HSC generation are incomplete. Many signaling pathways are known to be critical for the successful generation of HSCs in vivo, however a role for Pdgfr signaling better known for a role in vascular tissues, has not been investigated. We treated embryos at different developmental temporal windows with a pharmacological inhibitor for tyrosine kinase activity of Pdgfr. Surprisingly we found that Pdgfr signaling is required for HSCs well before the formation of HSCs during early somitogenesis, later Pdgfr signaling is required for intersegmental vessel sprouting but is dispensable for HSC formation as previously reported. The loss of HSCs during this early drug treatment window accompanied by defects to dorsal and somitic tissues, suggest that these phenotypes may be related. Formation of dorsal aorta, sclerotome, and HSCs is a Notch-signaling dependent process, we therefore asked if Pdgfr signaling was related to Notch signaling. We found that activation of Notch signaling in the endothelium was inhibited in the context of Pdgfr inhibition, furthermore we found that ectopic activation of Notch signaling rescued HSCs in these embryos suggesting a novel pathway by which Pdgfr signaling is involved in HSC specification.
Abstract Title : Role of Obscurin for Cardiac Calcium Handling
Abstract : The sarcoplasmic reticulum (SR) is responsible for the storage of calcium in skeletal and cardiac muscle cells. Calcium is responsible for muscle contraction and acts as one of the most important second-messenger molecules through modulation of multiple signaling pathways. Loss of control over calcium handling is often associated with cardiac diseases, like cardiac arrhythmias and heart failure (1,2). One of the proteins that is important for the organization and function of the SR in cross striated muscle cells is the giant muscle specific protein obscurin (3). We hypothesized that lack of obscurin in cardiac muscle has an impact on calcium-handling and excitation-contraction coupling. To investigate our hypothesis we analyzed changes to neonatal and adult cardiomyocytes from control and obscurin knockout mice using the calcium-sensitive dyes fluo-4 and fura-2. We discovered for neonatal cardiomyocytes that the time-to-peak values for calcium sparks is significantly increased in obscurin knockout cells compared to controls, indicating that lack of obscurin leads to an increase in calcium leakage from the SR. We further investigated calcium transients of adult cardiomyocytes and recorded significant changes to several parameters, including peak calcium amplitude, transient-rise and -return velocities as well as peak cell shortening. Taken together, these data indicate that obscurin plays important roles for proper SR calcium-handling and excitation-contraction. 1. Neef S, Maier LS. Novel aspects of excitation-contraction coupling in heart failure. Basic Res Cardiol. 2013;108(4):360. 2. Bers DM, Shannon TR. Calcium movements inside the sarcoplasmic reticulum of cardiac myocytes. J Mol Cell Cardiol. 2013 May;58:59-66. 3. Lange S*, Ouyang K*, Meyer G, Cui L, Cheng H, Lieber RL, Chen J. Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum. Journal of Cell Science. 2009.122(Pt 15):2640-50.
Abstract Title : Homolog Avoidance (Tiling) Requires the Calcium-Activated Protease Calpain for Process Retraction.
Abstract : Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech such as the AP cells, undergo a form of contact-dependent homolog avoidance, a form of cellular tiling behavior. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994; J Neurosci. 14). However, early deletion of an AP neuron or selective knockdown of the gap junction gene Inx1 (Baker at al., 2013, J Neurosci. 33), leads to resumed growth and permanent maintenance of the projections of neighboring APs. Continued growth was also observed when a closed-channel mutant of Inx1 was expressed by the AP neuron indicating the likely exchange of a soluble signal between the cells. Here we explore the nature of the signal that causes the AP?s connective processes to retract. Following the formation of the transient GJ, the distal processes of the AP neuron were observed to undergo Wallerian fragmentation, suggesting the possible activation of an intracellular protease. Calcium imaging has also revealed that these distal GJs permit calcium exchange between the contacting processes suggesting that one potential candidate for mediation may be the calcium-activated protease calpain which has been shown to function downstream of calcium transients. Accordingly, we employed RNAi to knockdown a neuronal leech calpain (CalI) in individual AP neurons in the developing embryo and found that homolog avoidance was significantly impaired. AP neurons were observed to extend processes into and beyond their adjacent ganglia, mimicking the effects of cellular ablation or Inx1 knockdown. Ongoing studies will explore the possible link between GJ signaling and CalI activation by targeting the source of compartmental calcium transients by examining the role-played by voltage-gated calcium channels and internal stored release mechanisms.