Browsing by Subject "Cell biology"

Now showing 1 - 4 of 4

Coordinated structural plasticity across synapses in the adult hippocampus
(2015-05) Chirillo, Michael August; Harris, Kristen M.; Bear, Mark F.; Colgin, Laura L.; Golding, Nace L.; Raab-Graham, Kimberly F.
Neural circuitry is determined primarily by trillions of synaptic junctions that link cells in the nervous system. Understanding how the structure of the synapse influences its function has been a central goal of cellular neuroscience since synapses were first recognized more than a century ago. Long-term potentiation (LTP), a long lasting enhancement of synaptic efficacy, is a well-characterized cellular correlate of learning and memory that results in dramatic structural remodeling of the synapse. Research has focused heavily on the postsynaptic structural remodeling that occurs to support LTP, but concomitant presynaptic and subcellular remodeling during LTP has been left largely unexplored. To address these questions, three-dimensional reconstructions from serial section electron microscopy of presynaptic boutons, vesicle pools, and dendritic smooth endoplasmic reticulum (SER) in hippocampal area CA1 were created and quantified. The data presented in this dissertation demonstrate that coordinated structural plasticity occurs at both pre- and postsynaptic sides of adult hippocampal synapses by 2 hours during LTP induced with theta burst stimulation. Presynaptically, the number of presynaptic boutons correlated perfectly with fewer dendritic spines during LTP that were previously reported, suggesting that synaptic units act as cohesive structures. Vesicle pools were mobilized and vesicle transport packets were moved into boutons or were released in transit. Dendritic SER is a ubiquitous intracellular membranous network involved in calcium signaling and protein modification. The complexity of SER influences the movement of diffusible membrane cargo. SER was dramatically remodeled during LTP, redistributing from the shaft of the dendrite into spines and becoming highly complex near synapses that were largest during LTP. As a preliminary investigation into how normal mechanisms of structural plasticity described in this dissertation might go awry under conditions of synaptic pathology, three-dimensional reconstructions of CA1 synaptic ultrastructure in a mouse model of Fragile X, which is known to express exaggerated mGluR-dependent long-term depression (LTD), were created and quantified. Synaptic ultrastructure was similar with that of the wild-type mouse, suggesting that structural malformation in FX might be confined to development or to other brain regions.
Dynamic micro-3D-printed substrates for characterizing cellular responses to topography
(2014-08) Ali, Maryam; Shear, Jason B.
Cell cultures provide researchers the opportunity to observe cell behavior in response to specific, well-defined environmental cues, leading to insights that enable better engineering design for tissue culture and other biomedical applications. Chemical and electrical stimuli have been successfully applied to cultured cells to approximate aspects of the dynamic conditions experienced in vivo. However, in vitro topographical cues have mostly been limited to static substrates that do not subject cells to the dynamic conditions they experience in vivo when tissue remodels during development and wound healing. Delivering dynamic topographical cues to cultured cells can answer long-standing questions about mechanisms of cell morphology changes. Such capabilities could also facilitate engineering of wound-healing matrices and nerve guidance conduits by promoting migration of cells and providing directional guidance to cellular processes. This dissertation describes the development of approaches for introducing in situ topographical cues to cell cultures and inducing responses such as neurite guidance and cell alignment. Both strategies undertaken in this work make use of multiphoton-promoted photochemistry to print and manipulate three-dimensional microscopic protein hydrogel structures. In one approach, a technique referred to as micro-3D printing, topographical guidance cues are printed in the proximity of cultured cells to guide the growth of cellular processes. By translating a tightly-focused pulsed laser beam through a printing reagent solution flooding cultured cells, features are printed that provide physical guidance to extending neurites from NG108-15 cells, a neuronal model cell type. In another approach, an innovative technique known as micro-3D imprinting is developed for producing micrometer-scale depressions on the surfaces of photoresponsive protein hydrogels. The impact of various experimental parameters on topographical feature dimensions is characterized. Micro-3D imprinting is used to introduce dynamic topographical changes on a cell culture substrate, demonstrating that NIH-3T3 cells, a fibroblast cell model, alter their morphology and alignment in response to the introduction of a grooved surface topography. This set of approaches introduces new tools to the repertoire of cell biologists for exploring the behavior of cells growing in a spatio-temporally dynamic environment, opening possibilities for studies of cellular behavior in conditions that may better reflect environments cells experience in vivo.
One cell as a mixture : simulation of the mechanical responses of valve interstitial cells
(2016-08) Sakamoto, Yusuke; Sacks, Michael S.; Prudhomme, Serge; Gonzalez, Oscar; Ghattas, Omar; Rodin, Gregory J; Guilak, Farshid
The function of the heart valve interstitial cells (VICs) are intimately connected to heart valve tissue remodeling and repair as well as initiation of pathological processes. It is known that excessive and persisting environmental changes cause the improper regulations of VICs, and a clinically significant valve pathology may result. Much of VIC function is modulated through changes in stress fiber activation, resulting in part from changes in external loading by the surrounding extracellular matrix (ECM) and cytokines. Thus, current research challenges aim at characterizing the mechanisms that activate VIC contractility, and at modeling the mechanical interactions of contractile VICs with the surrounding valve matrix. Especially, many questions remain as how stress fibers develop active contractile forces under varying normal and pathological conditions. The main objective of this dissertation is to develop a novel computational model of a VIC capable of describing its mechanical response under different external stimuli and activation states. To this end, solid mixture model framework of a VIC is developed, where the VIC cytoplasm is treated as a solid mixture of two phases: isotropic cytoskeleton and stress fibers with some orientations. The stress fiber model is then incrementally extended to capture more and more complex mechanical responses of VICs. The finite element simulations are performed with the aid of experimental data to investigate how the internal mechanics of VICs, such as solid cytoskeletal network, contracting stress fibers, and cell nucleus, affect the mechanical responses of VICs within a native tissue. The development of the computational model of a VIC as well as its numerical implementation are critical to study the heart valve disease in cellular level because of the complexity of the mechanisms and difficulty of directly analyzing the subcellular mechanics. The computational model in conjunction with experimental data provide insight into how the VICs respond within the native valve tissue, and how the heart valve disease may initiate. This dissertation is the first step towards developing prevention mechanisms and cure for the heart valve disease from cellular and subcellular levels.
Protein trafficking within ciliated cells
(2021-08-13) Hibbard, Jaime; Wallingford, John B.; Dickinson, Daniel; Marcotte, Edward; Finkelstein, Ilya; Kim, Jonghwan
Cilia are microtubule-based organelles that are crucial for embryonic development and tissue homeostasis post-development. Cilia are composed of thousands of proteins that must be trafficked from their site of synthesis in the cell body. Active transport of protein cargoes within cilia is mediated by the intraflagellar transport complex (IFT). IFT trafficking within cilia is well studied, but the role of IFT proteins in the cytoplasm is less understood. In my graduate work, I investigated modes of protein trafficking within ciliated cells. In Chapter 1, I introduce the routes by which ciliary proteins can traffic from their site of synthesis to the basal body. In Chapter 2, I describe my work studying the mechanism of IFT trafficking from the cytoplasm to the basal body. Finally, in Chapter 3, I introduce a pipeline to identify protein cargoes of IFT. This work provides new insights into protein trafficking within ciliated cells, which is crucial for ciliary signaling.