Browsing by Author "Kamba, Tisianna K."

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Artificial Intelligence Approaches to Assessing Primary Cilia
    (MyJove Corp., 2021-05-01) Bansal, Ruchi; Engle, Staci E.; Kamba, Tisianna K.; Brewer, Kathryn M.; Lewis, Wesley R.; Berbari, Nicolas F.; Biology, School of Science
    Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.
  • Thumbnail Image
    Item
    Artificial Intelligence Approaches to Assessing Primary Cilia
    (MyJove Corporation, 2021-05-01) Bansal, Ruchi; Engle, Staci E.; Kamba, Tisianna K.; Brewer, Kathryn M.; Lewis, Wesley R.; Berbari, Nicolas F.; Biology, School of Science
    Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.
  • Thumbnail Image
    Item
    Assessing neuronal ciliary localization of Melanin Concentrating Hormone Receptor 1 in vivo
    (2021-08) Kamba, Tisianna K.; Berbari, Nicolas F.; Mastracci, Teresa; Dai, Guoli
    Obesity is a growing pandemic that claims close to three hundred thousand lives per year in the United States alone. Despite strong interest and investment in potential treatments, obesity remains a complex and challenging disorder. In the study of obesity, mouse models have been excellent tools that help in understanding the function of different genes that contribute to this disease of energy homeostasis. However, it was surprising when disfunction in primary cilia was found to be linked to syndromic obesity. To understand the role of primary cilia in obesity, a growing subset of GPCRs have been identified to selectively localize to the organelle. Several of which have known roles in energy homeostasis. In some examples, ciliary GPCRs appear to dynamically localize to the organelle; such is the case of GPR161 and smoothened in the hedgehog signaling pathway. Thus, we were interested to see if other GPCRs dynamically localize to the primary cilia as part of their regulation of energy homeostasis. For example, the GPCR MCHR1 selectively localizes to the cilia and is involved in energy homeostasis. Although much is known about the expression of the receptor in the brain, how its ciliary subcellular localization impacts its roles in energy homeostasis is unknown. Observing neuronal cilia in vivo remains a difficult task as some of the available tools such as tagged alleles rely on overexpression of ciliary protein which may impact function. Additionally, most of the work is done in vitro, leaving much to be discovered about neuronal cilia in vivo. In this thesis, we show that using a newly constructed reporter allele mCherryMCHR1, we can see ciliary expression of MCHR1 in the brain of developing and adult mice; more specifically in the ARC and PVN. Subsequently, using a novel Artificial intelligence analysis approach, we measured the length and composition of MCHR1 positive cilia under physiological conditions associated with MCHR1 function. Although in this work we are reporting no changes in dynamic localization of MCHR1 in the hypothalamus specifically, we are not excluding the potential for changes in other regions of the brain or under other conditions; and we are suggesting that pharmacological approaches may help highlight potential ciliary GPCR dynamic localization.
  • Thumbnail Image
    Item
    An N‐terminal fusion allele to study melanin concentrating hormone receptor 1
    (Wiley, 2021-08) Jasso, Kalene R.; Kamba, Tisianna K.; Zimmerman, Arthur D.; Bansal, Ruchi; Engle, Staci E.; Everett, Thomas; Wu, Chang-Hung; Kulaga, Heather; Reed, Randal R.; Berbari, Nicolas F.; McIntyre, Jeremy C.; Biology, School of Science
    Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.