Repository logo

Browsing by Author "Krapf, Diego, advisor"

Now showing 1 - 9 of 9
  • Thumbnail Image
    ItemEmbargo
    3D localization of cytoskeleton in mouse spermatids using stochastic optical reconstruction microscopy
    (Colorado State University. Libraries, 2022) Sunny, Reshma, author; Krapf, Diego, advisor; Nikdast, Mahdi, committee member; Prasad, Ashok, committee member
    It is estimated by the World Health Organization that globally 186 million individuals live with infertility. Studies have shown that cause of male infertility is unknown in 30 to 50% of the cases. Over the last several years teratozoospermias have been investigated and have been backtracked to events in spermatogenesis. The development of the acrosome and the manchette, protein and vesicle transport in spermatids, and sperm head shaping are crucial steps in the formation of healthy sperms. The cytoskeleton in spermatids plays a crucial role in shaping the sperm head. The acroplaxome exerts forces on the nucleus and gives the mammalian sperm head its species-specific shape, and also facilitates the proper attachment of the nuclear cap called the acrosome, containing the enzymes required for sperm penetration of the oocyte. The manchette should be intact and formed properly to have shortened diameter as spermatids differentiate so that it can constrict the base of the nucleus to shape the head, and also facilitate the transport of cargo to the base of the cell. Thus as studies have confirmed, the disruption in the organization of the cytoskeleton is a concern for infertility. Hence it is crucial to learn more about the cytoskeletal structures in spermatids. The goal of this thesis is to 3D localize these structures. The major structures we are interested in are the acroplaxome and the manchette. For this, we use a super-resolution microscopy method called Stochastic Optical Reconstruction Microscopy to image spermatid cytoskeleton. Our experiments confirmed the presence of α-tubulin in the manchette and that of F-actin in the manchette and the acroplaxome, as previously observed by researchers with 2D confocal images. We observed that the manchette reduces in diameter and progresses to the caudal portion of the cell at the later steps of differentiation and that the structure forms completely at step 10 and disassembles after step 14.
  • Thumbnail Image
    ItemEmbargo
    Anomalous diffusion of mRNA in the cytoplasm of HeLa cells
    (Colorado State University. Libraries, 2024) Roessler, Ryan, author; Krapf, Diego, advisor; Stasevich, Tim, committee member; Prasad, Ashok, committee member
    Information about the diffusive motion of RNA would provide insights into intracellular structures and functions, as well as gene expression and genetic regulation. We study the motion of individual messenger RNA molecules in the cytoplasm of HeLa cells. RNAs are imaged in live cells via total internal reflection (TIRF) microscopy. In order to visualize individual RNA molecules expressing the MYH9 gene, they were labeled via MS2 stem loops bound to coat proteins tagged with the HaloTag-JF646 fluorophore. We then used single-particle tracking to obtain trajectories of individual molecules. Trajectories were analyzed in terms of their mean-squared displacement (MSD) and power spectral density (PSD). We observed non-ergodic, subdiffusive behavior, with statistics that depend on observation time, i.e., aging. Additionally, we observe stochastic switching between two mobility states with an order of magnitude difference in diffusivity. This switching process is responsible for the aging nature of the system. When compared to the cytoplasmic motion of synthetic nanoparticles, the analysis of RNA trajectories gives rise to discrepancies that raise questions about specific intracellular interactions.
  • Thumbnail Image
    ItemOpen Access
    Atomic force microscopy: more than surface imaging
    (Colorado State University. Libraries, 2020) Bishop, Terrance Tyler, author; Krapf, Diego, advisor; Prasad, Ashok, committee member; Van Orden, Alan, committee member
    Atomic Force Microscopy (AFM) is a powerful imaging tool that has capabilities that go beyond the abilities of most other microscopes. Here, three examples of these capabilities were considered. First, the AFM was operated in an image generating mode to determine the surface heterogeneity of polysaccharide membranes. Second, the AFM was used to record force-indentation curves, these curves were fit with a Hertzian model to determine the stiffness of murine smooth muscle cells. Finally a approach for attaching 10 µm and 2 µm polystyrene beads to tip-less AFM cantilevers was proposed, and a viscoelastic contact model was tested to determine the viability of the created probes.
  • Thumbnail Image
    ItemOpen Access
    Characterizing the diffusional behavior and trafficking pathways of Kv2.1 using single particle tracking in live cells
    (Colorado State University. Libraries, 2013) Weigel, Aubrey, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Bamburg, James, committee member; Bartels, Randy, committee member
    Studying the diffusion pattern of membrane components yields valuable information regarding membrane structure, organization, and dynamics. Single particle tracking serves as an excellent tool to probe these events. We are investigating of the dynamics of the voltage gated potassium channel, Kv2.1. Kv2.1 uniquely localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. The work reported here examines the diffusion pattern of Kv2.1 and determines alternate functional roles of surface clusters by investigating recycling pathways using single particle tracking in live cells. The movement of Kv2.1 on the cell surface is found to be best modeled by the combination of a stationary and non-stationary process, namely a continuous time random walk in a fractal geometry. Kv2.1 surface structures are shown to be specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells. Both Kv2.1 and Kv1.4, a non-clustering membrane protein, are inserted and retrieved from the plasma membrane at the perimeter of Kv2.1 clusters. From the distribution of cluster sizes, using a Fokker-Planck formalism, we find there is no evidence of a feedback mechanism controlling Kv2.1 domain size on the cell surface. Interestingly, the sizes of Kv2.1 clusters are rather governed by fluctuations in the endocytic and exocytic machinery. Lastly, we pinpoint the mechanism responsible for inducing Kv2.1 non-ergodic dynamics: the capture of Kv2.1 into growing clathrin-coated pits via transient binding to pit proteins.
  • Thumbnail Image
    ItemOpen Access
    Compartmentalization of membrane proteins by the actin cytoskeleton
    (Colorado State University. Libraries, 2013) Higgins, Jenny, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Bamburg, James, committee member; Azimi-Sadjadi, Mahmood, committee member
    Acting as the point of contact for the outside world, the plasma membrane is crucial for cellular signaling events. Proper organization of membrane components is necessary to accomplish this task. Although a number of experiments have demonstrated the compartmentalization of lipids and proteins on the plasma membrane, direct observation of the mechanisms by which the organization occurs has been challenging, in part due to the imaging restrictions of a diffraction-limited system and the dynamic nature of the membrane compartmentalization. Using photoactivated localization microscopy (PALM), a superresolution technique, we have captured the dynamics of compartments formed by the cortical actin cytoskeleton. Live human embryonic kidney (HEK293) cells were imaged with a temporal resolution of 2 s and a spatial resolution of 40 nm. The actin cytoskeleton forms compartments with a mean area of 2.3±0.3 μm2 that are partially outlined by actin bundles. When the PALM images of actin were combined with single particle tracking of membrane proteins, we directly observed the cytoskeleton acting as a barrier to the diffusion of Kv2.1 and Kv1.4, two voltage-gated potassium channels. In addition, we used a novel compartment detection and tracking algorithm to show that Kv2.1 and Kv1.4 channels avoid actin when changing compartments. This work represents the first direct observations of individual membrane protein interactions with barriers formed by the actin cytoskeleton.
  • Thumbnail Image
    ItemOpen Access
    Force spectroscopy and dynamics in biological systems
    (Colorado State University. Libraries, 2019) Schroder, Bryce William, author; Krapf, Diego, advisor; Bark, David, committee member; Popat, Ketul, committee member; DeLuca, Jennifer, committee member
    Communication is key to any process involving the transmission of information or some sort of signal. For communication to occur, a signal must be created that can be detected. Cells communicate through cues transmitted in the forms of chemical and mechanical signals. The most fundamental means for transmitting chemical cues is through the process of diffusion. A single particle undergoing diffusion is considered to undergo Brownian motion, which can be modelled as a random walk. The random walk behavior is characteristic of both the particles properties and the fields in which it is occurring. An unbiased walk will be completely random without outside influence. A biased walk will be random within the confines of a potential influencing its direction. Both are Stochastic processes characterized through probabilistic models with known solutions. The work herein presents the development of single molecule experiments and the associated particle tracking tools targeting particles undergoing biased random walks within a trapping potential on or near a cellular membrane. In the first set of experiments, the trapping potential, an optical tweezers setup, has been developed and employed in measuring cellular membrane biophysical properties as well as blebbing forces. The optical trap was also used to directly measure flow driven forces in live embryonic zebrafish, the first known measurements of this kind. In the second set of experiments, synthetic lipid bilayers provided a trapping potential in a single dimension for protein binding experiments leading to exchanges between free, 3-dimensional diffusion and bound, or biased, 2-dimensional diffusion. In all cases, stochastic models have been used in conjunction with image-based particle tracking tools to better characterize the biophysical properties and forces associated with the cellular membrane and its means of signal transduction. These measurements are key to understanding both the chemical and mechanical signaling means by which the cellular membrane transduces an external signal into an internal response.
  • Thumbnail Image
    ItemOpen Access
    Single molecule fluorescence measurements of complex systems
    (Colorado State University. Libraries, 2017) Sadegh, Sanaz, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Chong, Edwin, committee member; Prasad, Ashok, committee member
    Single molecule methods are powerful tools for investigating the properties of complex systems that are generally concealed by ensemble measurements. Here we use single molecule fluorescent measurements to study two different complex systems: 1/ƒ noise in quantum dots and diffusion of the membrane proteins in live cells. The power spectrum of quantum dot (QD) fluorescence exhibits 1/ƒ noise, related to the intermittency of these nanosystems. As in other systems exhibiting 1/ƒ noise, this power spectrum is not integrable at low frequencies, which appears to imply infinite total power. We report measurements of individual QDs that address this long-standing paradox. We find that the level of 1/ƒβ noise for QDs decays with the observation time. We show that the traditional description of the power spectrum with a single exponent is incomplete and three additional critical exponents characterize the dependence on experimental time. A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal.
  • Thumbnail Image
    ItemOpen Access
    Super-resolution imaging and modeling of murine sperm during capacitation process
    (Colorado State University. Libraries, 2019) Xu, Xinran, author; Krapf, Diego, advisor; Munsky, Brian, committee member; Pezeshki, Ali, committee member; Tamkun, Michael, committee member
    The effort to achieve better spatial resolution beyond the diffraction limit has been dedicated for many years. In the past decade, super-resolution microscopy methods have successfully advanced into extremely powerful tools to reveal hidden three-dimensional structures and properties in various biological complex systems. Here we use single-molecule localization based three-dimensional super-resolution microscopy to study the mouse sperm capacitation process, a critical step in gaining the fertilization ability. On top of that, we construct a stochastic model to represent this signaling pathway in order to be able to predict the cellular event within the capacitation. The major subjects we are interested in can be categorized into two parts: actin-based cytoskeleton and capacitation-associated signaling proteins. In the midpiece, we discovered that F-actin forms a highly specialized double helical structure, which has been the very first observation among species and has disappeared in the principal piece. Similarly, the distinctive compartments regarding actin-binding proteins have also been visualized in the mouse sperm tail. Additionally, the structure as well as localization of capacitation central mediator, protein kinase A have been investigated to address the significance of spatial positioning during the capacitation event. As the capacitation end point reporter, tyrosine phosphorylation localization has been studied to help identify its real upstream kinase among other candidates. As in many regulating processes, second messenger Ca2+ plays a vital role in the capacitation process, which needs to be conveyed by the sperm specific calcium channel CatSper. We show the structural relation of a small GTPase Cdc42 to CatSper, implying its key role in transporting Ca2+. Considering that major critical signaling molecules are well characterized in the complex capacitation network, we choose a different method–stochastic modeling, other than experimental studies, surpassing the need for probing the behavior of a large number of individual cells over time, to describe capacitation process and furthermore to predict the behavior of sperm. With the known pathways of those signaling molecules in hand, we are able to build a stochastic model by utilizing chemical master equations. A couple sets of experimental measurements are used to assist in quantifying the model.
  • Thumbnail Image
    ItemOpen Access
    The kinetics of proteins on lipid bilayers
    (Colorado State University. Libraries, 2017) Nepal, Kanti, author; Krapf, Diego, advisor; Peersen, Olve, committee member; Levinger, Nancy, committee member
    Signaling molecules trigger downstream signaling pathways when they arrive at the plasma membrane. They have to be recruited to the plasma membrane by membrane targeting domains. Our experiments throughout focus on understanding kinetics of C2 domain's diffusion on the membrane. In contrast to trans-membrane proteins, interactions between these domains and the plasma membrane is found to be peripheral and transient. These proteins perform two dimensional diffusion on membrane surfaces and faster three dimensional diffusion in the bulk. We label proteins at the single molecule level and do single particle tracking. In addition to two dimensional surface diffusion, it is sometimes observed that they dissociate from the membrane and rebind at a another location of the membrane after a short journey in the bulk solution. The time averaged mean square displacement (MSD) analysis of individual trajectories is linear whereas ensemble average MSD is superdiffusive. The distribution of displacements fit to a Gaussian distribution followed by a long tail which is Cauchy's distribution. This long tail in cauchy's distribution is from the larger displacements caused by jumps of molecule to explore greater area for efficient target search. The second section of this thesis explored the effect of crowding agents on these proteins. Polyethylene glycol (PEG) is used here to simulate crowded cellular environment aiming to understand its effect on membrane targeting C2 domains as well as on the lipid bilayer. In this chapter, we recognized that a crowding agent like PEG plays a significant role in changing the trend on diffusion behavior of C2 domains. When the PEG concentration is increased, there is a decrease in the transition of molecule between the surface and the bulk phase. With the same series of PEG concentration, there is increase in population of immobile C2 domains and desorption time. But no such increasing or decreasing trend is seen on the lipid bilayer alone. Experiments were reproduced and imaged a number of times using total internal reflection (TIRF) and fluorescence recovery after photobleaching (FRAP) techniques. Lastly, a small part of my thesis also dealt with set of experiments done to monitor tethered particle motion of DNA as well as flow extension experiments on DNA and RNA using bright field microscopy. DNA/RNA had beads tethered to one end of the strand and other end to the cover glass. Primary results are presented.