Browsing by Subject "Microtubules"
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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 ScienceCilia 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.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 ScienceCilia 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.Item Change in Cerebrospinal Fluid Tau Microtubule Binding Region Detects Symptom Onset, Cognitive Decline, Tangles, and Atrophy in Dominantly Inherited Alzheimer's Disease(Wiley, 2023) Horie, Kanta; Li, Yan; Barthélemy, Nicolas R.; Gordon, Brian; Hassenstab, Jason; Benzinger, Tammie L. S.; Fagan, Anne M.; Morris, John C.; Karch, Celeste M.; Xiong, Chengjie; Allegri, Ricardo; Mendez, Patricio Chrem; Ikeuchi, Takeshi; Kasuga, Kensaku; Noble, James; Farlow, Martin; Chhatwal, Jasmeer; Day, Gregory; Schofield, Peter R.; Masters, Colin L.; Levin, Johannes; Jucker, Mathias; Lee, Jae-Hong; Roh, Jee Hoon; Sato, Chihiro; Sachdev, Pallavi; Koyama, Akihiko; Reyderman, Larisa; Bateman, Randall J.; McDade, Eric; Dominantly Inherited Alzheimer Network; Neurology, School of MedicineObjective: Identifying cerebrospinal fluid measures of the microtubule binding region of tau (MTBR-tau) species that reflect tau aggregation could provide fluid biomarkers that track Alzheimer's disease related neurofibrillary tau pathological changes. We examined the cerebrospinal fluid (CSF) MTBR-tau species in dominantly inherited Alzheimer's disease (DIAD) mutation carriers to assess the association with Alzheimer's disease (AD) biomarkers and clinical symptoms. Methods: Cross-sectional and longitudinal CSF from 229 DIAD mutation carriers and 130 mutation non-carriers had sequential characterization of N-terminal/mid-domain phosphorylated tau (p-tau) followed by MTBR-tau species and tau positron emission tomography (tau PET), other soluble tau and amyloid biomarkers, comprehensive clinical and cognitive assessments, and brain magnetic resonance imaging of atrophy. Results: CSF MTBR-tau species located within the putative "border" region and one species corresponding to the "core" region of aggregates in neurofibrillary tangles (NFTs) increased during the presymptomatic stage and decreased during the symptomatic stage. The "border" MTBR-tau species were associated with amyloid pathology and CSF p-tau; whereas the "core" MTBR-tau species were associated stronger with tau PET and CSF measures of neurodegeneration. The ratio of the border to the core species provided a continuous measure of increasing amounts that tracked clinical progression and NFTs. Interpretation: Changes in CSF soluble MTBR-tau species preceded the onset of dementia, tau tangle increase, and atrophy in DIAD. The ratio of 4R-specific MTBR-tau (border) to the NFT (core) MTBR-tau species corresponds to the pathology of NFTs in DIAD and change with disease progression. The dynamics between different MTBR-tau species in the CSF may serve as a marker of tau-related disease progression and target engagement of anti-tau therapeutics.Item An in vitro study of the mechanisms that underlie changes in neuronal sensitivity and neurite morphology following treatment with microtubule targeting agents(2014-11) Pittman, Sherry Kathleen; Fehrenbacher, Jill C.; Cummins, Theodore R.; Hingtgen, Cynthia M.; Hudmon, Andrew; Vasko, Michael R.Microtubule targeting agents (MTAs) are chemotherapeutics commonly used in the treatment of breast, ovarian, lung, and lymphoma cancers. There are two main classes of MTAs based upon their effects on microtubule stability. The two classes are the destabilizing agents, which include the drug vincristine, and the stabilizing agents, which include paclitaxel and epothilone B. These drugs are highly effective antineoplastics, but their use is often accompanied by several side effects, one of which is peripheral neuropathy. Peripheral neuropathy can be characterized by burning pain, tingling, loss of proprioception, or numbness in the hands and feet. In some patients, the MTA-induced peripheral neuropathy is debilitating and dose-limiting; however, there are no effective prevention strategies or treatment options for peripheral neuropathy as the mechanisms mediating this side effect are unknown. The goal of this work was to investigate MTA-induced effects on neuronal activity and morphology in order to elucidate the underlying mechanisms involved in the development of MTA-induced peripheral neuropathy. As an indicator of sensory neuronal activity, the basal and stimulated release of the putative nociceptive peptide, calcitonin gene-related peptide (CGRP), was measured from sensory neurons in culture after exposure to the MTAs paclitaxel, epothilone B, and vincristine. Neurite length and branching were also measured in sensory neuronal cultures after treatment with these MTAs. The results described in this thesis demonstrate that MTAs alter the stimulated release of CGRP from sensory neurons in differential ways depending on the MTA agent employed, the CGRP evoking-stimulus used, the concentration of the MTA agent, the duration of exposure to the MTA agent, and the presence of NGF. It was also observed that MTA agents decrease neurite length and branching, independent of the concentration of NGF in the culture media. Thus, this thesis describes MTA-induced alterations of sensory neuronal sensitivity and neurite morphology and begins to elucidate the underlying mechanisms involved in MTA-induced alterations of sensory neurons. These findings will undoubtedly be used to help elucidate the mechanisms underlying MTA-induced peripheral neuropathy.Item Microtubule modifications and stability are altered by cilia perturbation and in cystic kidney disease(Wiley, 2013) Berbari, Nicolas F.; Sharma, Neeraj; Malarkey, Erik B.; Pieczynski, Jay N.; Boddu, Ravindra; Gaertig, Jacek; Guay-Woodford, Lisa; Yoder, Bradley K.; Biology, School of ScienceDisruption of the primary cilium is associated with a growing number of human diseases collectively termed ciliopathies. Ciliopathies present with a broad range of clinical features consistent with the near ubiquitous nature of the organelle and its role in diverse signaling pathways throughout development and adult homeostasis. The clinical features associated with cilia dysfunction can include such phenotypes as polycystic kidneys, skeletal abnormalities, blindness, anosmia, and obesity. Although the clinical relevance of the primary cilium is evident, the effects that cilia dysfunction has on the cell and how this contributes to disease remains poorly understood. Here, we show that loss of ciliogenesis genes such as Ift88 and Kif3a lead to increases in post-translational modifications on cytosolic microtubules. This effect was observed in cilia mutant kidney cells grown in vitro and in vivo in cystic kidneys. The hyper-acetylation of microtubules resulting from cilia loss is associated with both altered microtubule stability and increased α-tubulin acetyl-transferase activity. Intriguingly, the effect on microtubules was also evident in renal samples from patients with autosomal recessive polycystic kidneys. These findings indicate that altered microtubule post-translational modifications may influence some of the phenotypes observed in ciliopathies.Item Spatial regulation of MCAK promotes cell polarization and focal adhesion turnover to drive robust cell migration(American Society for Cell Biology, 2021-04-01) Zong, Hailing; Hazelbaker, Mark; Moe, Christina; Ems-McClung, Stephanie C.; Hu, Ke; Walczak, Claire E.; Biology, School of ScienceThe asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.Item Structure and dynamics of motor-driven microtubule bundles(Royal Society of Chemistry, 2024-07-24) Lemma, Bezia; Lemma, Linnea M.; Ems-McClung, Stephanie C.; Walczak, Claire E.; Dogic, Zvonimir; Needleman, Daniel J.; Medicine, School of MedicineConnecting the large-scale emergent behaviors of active cytoskeletal materials to the microscopic properties of their constituents is a challenge due to a lack of data on the multiscale dynamics and structure of such systems. We approach this problem by studying the impact of depletion attraction on bundles of microtubules and kinesin-14 molecular motors. For all depletant concentrations, kinesin-14 bundles generate comparable extensile dynamics. However, this invariable mesoscopic behavior masks the transition in the microscopic motion of microtubules. Specifically, with increasing attraction, we observe a transition from bi-directional sliding with extension to pure extension with no sliding. Small-angle X-ray scattering shows that the transition in microtubule dynamics is concurrent with a structural rearrangement of microtubules from an open hexagonal to a compressed rectangular lattice. These results demonstrate that bundles of microtubules and molecular motors can display the same mesoscopic extensile behaviors despite having different internal structures and microscopic dynamics. They provide essential information for developing multiscale models of active matter.Item A Tale of the Good and Bad: Remodeling of the Microtubule Network in the Brain by Cdk5(Springer, 2017-04) Shah, Kavita; Lahiri, Debomoy K.; Psychiatry, School of MedicineCdk5, a cyclin-dependent kinase family member, is a global orchestrator of neuronal cytoskeletal dynamics. During embryogenesis, Cdk5 is indispensable for brain development. In adults, it is essential for numerous neuronal processes, including higher cognitive functions such as learning and memory formation, drug addiction, pain signaling, and long-term behavior changes through long-term potentiation and long-term depression, all of which rely on rapid alterations in the cytoskeleton. Cdk5 activity becomes deregulated in various brain disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, attention-deficit hyperactivity disorder, epilepsy, schizophrenia, and ischemic stroke; these all result in profound remodeling of the neuronal cytoskeleton. This Commentary specifically focuses on the pleiotropic contribution of Cdk5 in regulating neuronal microtubule remodeling. Because the vast majority of the physiological substrates of Cdk5 are associated with the neuronal cytoskeleton, our emphasis is on the Cdk5 substrates, such as CRMP2, stathmin, drebrin, dixdc1, axin, MAP2, MAP1B, doublecortin, kinesin-5, and tau, that have allowed to unravel the molecular mechanisms through which Cdk5 exerts its divergent roles in regulating neuronal microtubule dynamics, both in healthy and disease states.